Great research starts with great data.

Learn More
More >
Patent Analysis of

Engineered enzyme having acetoacetyl-CoA hydrolase activity, microorganisms comprising same, and methods of using same

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10000744

Application Number

US15/023579

Application Date

23 September 2014

Publication Date

19 June 2018

Current Assignee

BRASKEM S.A.

Original Assignee (Applicant)

SLOVIC, AVRAM MICHAEL,BRASKEM S.A.

International Classification

C12N1/19,C12P7/04,C12N9/10,C12N9/16,C12N1/21

Cooperative Classification

C12N9/16,C12N9/13,C12P7/04,C12Y301/02011

Inventor

SLOVIC, AVRAM MICHAEL,GOUVEA, IURI ESTRADA,KOCH, DANIEL JOHANNES,GALZERANI, FELIPE

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 1 US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 2 US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 3
See all images <>

Abstract

The disclosure provides engineered enzymes that are capable of mediating the conversion of acetoacetyl-CoA to acetoacetate that do not react with the same order of magnitude with acetyl-CoA as they do with acetoacetyl-CoA (e.g., the engineered enzymes have a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity). Additionally, the disclosure provides modified microorganisms that comprise the engineered enzymes disclosed herein and methods of using same.

Read more

Claims

1. A modified microorganism comprising one or more polynucleotides encoding one or more enzymes in a pathway with acetoacetate as an intermediate or end-product, and an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity; and wherein the engineered enzyme comprises the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

2. The modified microorganism of claim 1, wherein the engineered enzyme comprises i) an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity and ii) a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1.

3. The modified microorganism of claim 2, wherein the enzyme having acetoacetyl-CoA transferase activity is from an enzyme family having 3-oxoacid CoA transferase activity.

4. The modified microorganism of claim 2, wherein the enzyme having acetoacetyl-CoA transferase activity is butyrate-acetoacetate CoA-transferase or acetoacetate-CoA transferase.

5. The modified microorganism of claim 2, wherein the enzyme having acetoacetyl-CoA transferase activity is from Clostridium acetobutylicum or Escherichia coli.

6. The modified microorganism of claim 1, wherein the engineered enzyme has a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.

7. The modified microorganism of claim 1, wherein the microorganism further comprises a disruption in one or more polynucleotides encoding one or more enzymes that decarboxylate pyruvate or a disruption in one or more polynucleotides encoding a transcription factor of an enzyme that decarboxylates pyruvate.

8. The modified microorganism of claim 7, wherein the disruption in the one or more polynucleotides encoding in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation.

9. The modified microorganism of claim 8, wherein the one or more enzymes that decarboxylate pyruvate include pyruvate decarboxylase 1 (pdc1), pdc5, and/or pdc6, and wherein the transcription factor of an enzyme that decarboxylates pyruvate is pdc2.

10. A method of culturing a microorganism, said method comprising: a.) providing a fermentable carbon source; and b.) culturing the modified microorganism of claim 9 in a fermentation media comprising the fermentable carbon source.

11. The method of claim 10, wherein the culturing is under anaerobic conditions.

Read more

Claim Tree

  • 1
    1. A modified microorganism comprising
    • one or more polynucleotides encoding one or more enzymes in a pathway with acetoacetate as an intermediate or end-product, and an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity
    • and wherein the engineered enzyme comprises the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
    • 2. The modified microorganism of claim 1, wherein
      • the engineered enzyme comprises
    • 6. The modified microorganism of claim 1, wherein
      • the engineered enzyme has a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.
    • 7. The modified microorganism of claim 1, wherein
      • the microorganism further comprises
  • 10
    10. A method of culturing a microorganism, said method comprising:
    • a.) providing a fermentable carbon source
    • and b.) culturing the modified microorganism of claim 9 in a fermentation media comprising the fermentable carbon source.
    • 11. The method of claim 10, wherein
      • the culturing is under anaerobic conditions.
See all independent claims <>

Description

BACKGROUND

The conversion of acetoacetyl-CoA to acetoacetate (FIG. 1) is an essential step in metabolic pathways with such intermediates. The specific hydrolysis of the thioester bond between coenzyme A (a thiol) and acetoacetate (an acyl group carrier) in acetoacetyl-CoA is an efficient way to produce the aforementioned conversion. Two classes of naturally occurring enzymes have been used to mediate such conversion including, CoA transferases (E.C. 2.8.3.-) and CoA-hydrolases (thioesterases) (E.C. 3.1.2.-). However, while acetoacetate-CoA transferases require the presence of a non-activated acid acting as CoA acceptor, the CoA-hydrolases (acyl-CoA thioesterases) described to act on acetoacetyl-CoA are unspecific in the sense that they react with the same order of magnitude with acetyl-CoA, the substrate required for acetoacetyl-CoA formation by the enzyme thiolase (E.C.2.3.1.9), thereby degrading the substrate for the acetoacetyl-CoA biosynthesis itself.

Therefore, there exists a need in the art for improved enzymes to mediate the conversion of acetoacetyl-CoA to acetoacetate.

SUMMARY

The present disclosure provides engineered enzymes that are capable of mediating the conversion of acetoacetyl-CoA to acetoacetate that do not react with the same order of magnitude with acetyl-CoA as they do with acetoacetyl-CoA (e.g., the engineered enzymes have a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity).

The present disclosure also provides an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme comprises i) an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity; and ii) a substitution of a glutamic acid residue (i.e., the catalytic glutamic acid residue) to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1, or a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 46 of SEQ ID NO: 3, or a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 333 of SEQ ID NO: 5.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is butyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoA transferase.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from Clostridium acetobutylicum.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has the amino acid sequence as set forth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from Escherichia coli.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has the amino acid sequence as set forth in SEQ ID NO: 3.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has the amino acid sequence as set forth in SEQ ID NO: 5.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.

The present disclosure also provides an engineered enzyme having the amino acid sequence as set forth in SEQ ID NO: 2.

The present disclosure also provides a modified microorganism comprising one or more polynucleotides coding for one or more enzymes in a pathway with acetoacetate as an intermediate or end-product, and an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more enzymes that decarboxylate pyruvate include pdc1, pdc 5, and/or pdc6, and wherein the one or more transcription factors of the one or more enzymes that decarboxylate pyruvate include pdc2.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme comprises i) an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity and ii) a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is butyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoA transferase.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from Clostridium acetobutylicum.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme has the amino acid sequence as set forth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from Escherichia coli.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has the amino acid sequence as set forth in SEQ ID NO: 4.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has the amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.

The present disclosure also provides a method of engineering an enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity, the method comprising: a) selecting an enzyme having acetoacetyl-CoA transferase activity, and b) substituting a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1 in the enzyme having acetoacetyl-CoA transferase activity to produce an engineered enzyme.

In some embodiments of each or any of the above or below mentioned embodiments, the substitution is introduced via site directed mutagenesis.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is butyrate-acetoacetate CoA-transferase.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme having acetoacetyl-CoA transferase activity is from Clostridium acetobutylicum or Escherichia coli.

In some embodiments of each or any of the above or below mentioned embodiments, the engineered enzyme has a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.

The present disclosure also provides a method of producing one or more products from a fermentable carbon source, said method comprising: a.) providing a fermentable carbon source; and b.) contacting the fermentable carbon source with the modified microorganism as disclosed herein in a fermentation media, wherein the microorganism produces one or more products from the fermentable carbon source.

In some embodiments of each or any of the above or below mentioned embodiments, the carbon source is contacted with the modified microorganism under anaerobic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.

FIG. 1 depicts a reaction scheme for the formation of acetoacetate through hydrolysis of acetoacetyl-CoA.

FIG. 2 depicts reaction schemes for metabolic pathways with the intermediates acetoacetyl-CoA and acetoacetate.

FIG. 3 depicts an alignment of 3-oxoacid CoA-transferases illustrating the identification and location of the active glutamic acid residue.

FIG. 4 depicts an exemplary pathway for the co-production of 1-propanol and 2-propanol, where 1-propanol is produced via a dihydroxyacetone-phosphate intermediate.

FIG. 5 depicts an exemplary pathway for the co-production of 1-propanol and 2-propanol, where 1-propanol is produced via a glyceraldehyde 3-phosphate.

DETAILED DESCRIPTION

The conversion of acetoacetyl-CoA to acetoacetate (FIG. 1) is an essential step in metabolic pathways with such intermediates including, for example, pathways for the production of 3-hydroxy-butyrate, acetone or isopropanol (FIG. 2). However, no acetoacetyl-CoA specific hydrolase is known that can produce acetoacetate and regenerate free CoA without degrading acetyl-CoA, the substrate for the acetoacetyl-CoA biosynthesis itself. The present disclosure provides the rational engineering of a 3-oxoacid CoA-transferase with acetoacetyl-CoA substrate specificity (e.g., a butyrate-acetoacetate CoA-transferase—SEQ ID NO: 1; a acetate-acetoacetate CoA-transferase—SEQ ID NO: 2; or Acetate CoA-transferase—SEQ ID NO: 3) to an acetoacetyl-CoA specific hydrolase and its use in metabolic pathways utilizing acetoacetate as an intermediate or an end-product including, for example, pathways for the synthesis of 3-hydroxy-butyrate, acetone and/or isopropanol. The engineered enzyme has a higher activity on acetoacetyl-CoA versus acetyl-CoA (e.g., 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, or more). The engineered enzyme may comprise: i) an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity and ii) a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1, or a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 46 of SEQ ID NO: 3, or a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 333 of SEQ ID NO: 5; and have a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity. Exemplary 3-oxoacid CoA-transferases are listed in FIG. 3 as well as the location of the active glutamic acid residue that may be substituted to an aspartic acid residue to engineer an enzyme having acetoacetyl-CoA specific hydrolase activity.

The engineered acetoacetyl-CoA hydrolase disclosed herein solves the problem that no acetoacetyl-CoA specific hydrolase is known that can produce acetoacetate and regenerate free CoA. Natural wild type hydrolases are known to accept several acid CoA compounds with similar activities and can be expected to be very difficult to be engineered for such specificity. Thus, naturally occurring and known enzymes with acetoacetyl-CoA hydrolase activity suggested previously (see, US 2010/0261237 A1) create the problem of unspecific acid-CoA (e.g., acetyl-CoA) hydrolase activity. Such enzymes destroy the precursor necessary for the formation of their own substrate, i.e. acetoacetyl-CoA generation from two acetyl-CoA by thiolase enzyme. As a result, their use in metabolic pathways containing further acid-CoA intermediates is highly inefficient.

Additionally, the engineered acetoacetyl-CoA hydrolase disclosed herein solves the problem of requiring an acceptor molecule and processing another acid-CoA intermediate. Appropriate transferase enzymes for the enzymatic removal of the CoA group from acetoacetyl-CoA are typically specific. However, the reaction requires an acceptor acid molecule and yields a further acid-CoA compound that needs to be processed for regeneration of free CoA. Removing the necessity of an acceptor molecule enables the creation of simplified, usually more efficient pathways.

Furthermore, transferases already described to accept acetoacetyl-CoA as substrate have a Km value for the acceptor molecule that is above 10 mM which is about 1000 times higher than the Km value for the acetoacetyl-CoA donor substrate. So the acceptor concentration is a limiting factor of the transferase reaction. Utilizing an acetoacetyl-CoA hydrolase engineered from an acetoacetyl-CoA transferase (i.e., a 3-oxoacid CoA-transferase that accepts Acetoacetyl-CoA as a CoA donor) has the added benefit of a very low Km for the substrate acetoacetyl-CoA. This allows hydrolysis of acetoacetyl-CoA with a high reaction rate at low substrate concentrations and therefore can prevent accumulation of acetoacetyl-CoA and establish a “pull” on the preceding, thiolase mediated reversible acetoacetyl-CoA biosynthesis reaction. Since the thiolase reaction often represents a rate limiting step in a biosynthesis, such a pull can be highly beneficial for the performance of the entire appropriate metabolic pathway.

The invention disclosed herein has particular importance in the context of a microorganism such as Saccharomyces cerevisiase strain that has the pyruvate decarbolylase genes (e.g., PDC1, PDC5 and PDC6) disrupted and/or deleted. In this strain, the reaction catalyzed by this enzyme, namely the conversion of pyruvate to acetaldehyde and CO2 does not occur. The result of such a deletion is that acetaldehyde cannot be further reduced by alcohol dehydrogenase to make ethanol, and thus such strain is deemed ethanol null. A secondary effect of such a deletion is that such a strain also does not produce acetic acid, which in the 2-propanol pathway described herein (see, e.g., Table 3 and FIGS. 4 and 5), is an essential receptor for a CoA which is transferred from acetoacetyl-CoA as it is converted to acetoacetate by a transferase. Thus, in the absence of a CoA receptor for such a reaction, it is impossible to remove the CoA from acetoacetyl-CoA, and the pathway cannot advance to 2-propanol. Pyruvate decarbolylase null yeast strains modified to produce 2-propanol thus require either exogenous acetate to receive the CoA from acetoacetyl-CoA, or require the activity of an enzyme such as a hydrolase to remove such a CoA from acetoacetyl-CoA. The hydrolase thus proposed has practical application in the context of such a strain which is unable to produce acetic acid, but requires a manner to convert acetoacetyl-CoA to acetoacetate.

Microorganisms disclosed herein with an engineered acetoacetyl-CoA specific hydrolase may also be modified to have a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate. In an embodiment, the disruption in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation. In a further embodiment, the one or more enzymes that decarboxylate pyruvate include pdc1, pdc 5, and/or pdc6, and the one or more transcription factors of the one or more enzymes that decarboxylate pyruvate include pdc2. The microorganism may additionally comprise one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 1-propanol and/or 2-propanol from a fermentable carbon source under anaerobic conditions.

As used herein, the term “biological activity” or “functional activity,” when referring to a protein, polypeptide or peptide, may mean that the protein, polypeptide or peptide exhibits a functionality or property that is useful as relating to some biological process, pathway or reaction. Biological or functional activity can refer to, for example, an ability to interact or associate with (e.g., bind to) another polypeptide or molecule, or it can refer to an ability to catalyze or regulate the interaction of other proteins or molecules (e.g., enzymatic reactions).

As used herein, the term “culturing” may refer to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or on solid medium.

As used herein, the term “derived from” may encompass the terms originated from, obtained from, obtainable from, isolated from, and created from, and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to the another specified material.

As used herein, “exogenous polynucleotide” refers to any deoxyribonucleic acid that originates outside of the microorganism.

As used herein, the term “an expression vector” may refer to a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as a DNA coding sequence (e.g. gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host. Such control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, cosmid, phage particle, bacterial artificial chromosome, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome (e.g., independent vector or plasmid), or may, in some instances, integrate into the genome itself (e.g., integrated vector). The plasmid is the most commonly used form of expression vector. However, the disclosure is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.

As used herein, the term “expression” may refer to the process by which a polypeptide is produced based on a nucleic acid sequence encoding the polypeptides (e.g., a gene). The process includes both transcription and translation.

As used herein, the term “gene” may refer to a DNA segment that is involved in producing a polypeptide or protein (e.g., fusion protein) and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, the term “heterologous,” with reference to a nucleic acid, polynucleotide, protein or peptide, may refer to a nucleic acid, polynucleotide, protein or peptide that does not naturally occur in a specified cell, e.g., a host cell. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes. In contrast, the term homologous, with reference to a nucleic acid, polynucleotide, protein or peptide, refers to a nucleic acid, polynucleotide, protein or peptide that occurs naturally in the cell.

As used herein, the term a “host cell” may refer to a cell or cell line, including a cell such as a microorganism which a recombinant expression vector may be transfected for expression of a polypeptide or protein (e.g., fusion protein). Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell may include cells transfected or transformed in vivo with an expression vector.

As used herein, the term “introduced,” in the context of inserting a nucleic acid sequence or a polynucleotide sequence into a cell, may include transfection, transformation, or transduction and refers to the incorporation of a nucleic acid sequence or polynucleotide sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence or polynucleotide sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

As used herein, the term “non-naturally occurring” or “modified” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Non-naturally occurring microbial organisms of the disclosure can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

As used herein, the term “operably linked” may refer to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect. For example, a promoter may be operably linked to a coding sequence if it controls the transcription of the coding sequence.

As used herein, “1-propanol” is intended to mean n-propanol with a general formula CH3CH2CH2OH (CAS number—71-23-8).

As used herein, “2-propanol” is intended to mean isopropyl alcohol with a general formula CH3CH3CHOH (CAS number—67-63-0).

As used herein, the term “a promoter” may refer to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. A promoter may be an inducible promoter or a constitutive promoter. An inducible promoter is a promoter that is active under environmental or developmental regulatory conditions.

As used herein, the term “a polynucleotide” or “nucleic acid sequence” may refer to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Such polynucleotides or nucleic acid sequences may encode amino acids (e.g., polypeptides or proteins such as fusion proteins). Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present disclosure encompasses polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2′-O-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms polynucleotide, nucleic acid, and oligonucleotide are used herein interchangeably. Polynucleotides may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR′, COCH2 (formacetal), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.

As used herein, the term a “protein” or “polypeptide” may refer to a composition comprised of amino acids and recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms protein and polypeptide are used interchangeably herein to refer to polymers of amino acids of any length, including those comprising linked (e.g., fused) peptides/polypeptides (e.g., fusion proteins). The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

As used herein, related proteins, polypeptides or peptides may encompass variant proteins, polypeptides or peptides. Variant proteins, polypeptides or peptides differ from a parent protein, polypeptide or peptide and/or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids. Alternatively or additionally, variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL (see, infra). For example, variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with a reference sequence.

As used herein, the term “recovered,”“isolated,”“purified,” and “separated” may refer to a material (e.g., a protein, peptide, nucleic acid, polynucleotide or cell) that is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

As used herein, the term “recombinant” may refer to nucleic acid sequences or polynucleotides, polypeptides or proteins, and cells based thereon, that have been manipulated by man such that they are not the same as nucleic acids, polypeptides, and cells as found in nature. Recombinant may also refer to genetic material (e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another coding sequence or gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at decreased or elevated levels, expressing a gene conditionally or constitutively in manners different from its natural expression profile, and the like.

As used herein, the term “selective marker” or “selectable marker” may refer to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid sequence, polynucleotide or vector. Examples of selectable markers include but are not limited to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.

As used herein, the term “substantially anaerobic” means that growth of the modified microorganism takes place in culture media that comprises a dissolved oxygen concentration of less than 5 ppm.

As used herein, the term “substantially similar” and “substantially identical” in the context of at least two nucleic acids, polynucleotides, proteins or polypeptides may mean that a nucleic acid, polynucleotide, protein or polypeptide comprises a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) nucleic acid, polynucleotide, protein or polypeptide. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl. Acad. Sci. 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448.) In some embodiments, substantially identical polypeptides differ only by one or more conservative amino acid substitutions. In some embodiments, substantially identical polypeptides are immunologically cross-reactive. In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

As used herein, the term “transfection” or “transformation” may refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell. The exogenous nucleic acid or polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome. The term transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.

As used herein, the term “transformed,”“stably transformed,” and “transgenic” may refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

As used herein, the term “vector” may refer to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, single and double stranded cassettes and the like.

As used herein, the term “wild-type,”“native,” or “naturally-occurring” proteins may refer to those proteins found in nature. The terms wild-type sequence refers to an amino acid or nucleic acid sequence that is found in nature or naturally occurring. In some embodiments, a wild-type sequence is the starting point of a protein engineering project, for example, production of variant proteins.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure. Further, it will be understood that any of the substrates disclosed in any of the pathways herein may alternatively include the anion or the cation of the substrate.

Numeric ranges provided herein are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the disclosure, and is not intended to limit the disclosure to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the disclosure in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that can be formed by dividing a disclosed numeric value into any other disclosed numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the numerical values presented herein and in all instances such ratios, ranges, and ranges of ratios represent various embodiments of the present disclosure.

Engineering of Acetoacetyl-CoA Hydrolase

A transferase with acetoacetyl-CoA substrate specificity may be engineered to produce an acetoacetyl-CoA specific hydrolase. The disclosure contemplates that any method known in the art may be used to modify a transferase with acetoacetyl-CoA substrate specificity including, for example, site directed mutagenesis. In an embodiment, the transferase with acetoacetyl-CoA substrate specificity may be modified to comprise a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1. The engineered enzyme may be subjected to further mutagenesis (e.g., random mutagenesis) to further increase its hydrolase activity.

The present disclosure also provides a method of engineering an enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity, the method comprising: a) selecting an enzyme having acetoacetyl-CoA transferase activity, and b) substituting a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1 in the enzyme having acetoacetyl-CoA transferase activity to produce an engineered enzyme.

In an embodiment of the disclosure, an active site glutamate residue at position 51 of SEQ ID NO: 1, or an active site glutamic acid residue at position 46 of SEQ ID NO: 3, or an active site glutamic acid residue at position 333 of SEQ ID NO: 5, is substituted with an aspartate residue using site direct mutagenesis to generate a CoA hydrolase (i.e., a thioesterase) with higher activity on acetoacetyl-CoA versus acetyl-CoA.

Alternatively, in an embodiment of the disclosure an acetoacetyl-CoA hydrolase may be engineered from a transferase by directed evolution. In an exemplary method, libraries of at least partially random mutated acetoacetyl-CoA transferases are created and a mutant with the desired hydrolase activity is identified through appropriate screening and selection methods (i.e. detection of free CoA after contacting enzyme variant with acetoacetyl-CoA, but without acceptor molecule). Such a method can result in other mutations than an exchange of the active glutamate acid residue to result in hydrolase activity. For instance, the three dimensional structure of the protein could get changed in such a way, that the distance between substrate and acid group of the active glutamic acid residue is increased to the same extent as in a replacement of the active glutamic acid with an aspartic acid residue, with similar effects on enzyme activity.

It will be appreciated by one of skill in the art that the active site glutamate residue of an enzyme with acetoacetyl-CoA transferase activity can be readily identified in any known transferase (see, Table 1) by sequence alignment of such enzyme with SEQ ID NO: 1 and that any known transferase can be modified to produce an acetoacetyl-CoA specific hydrolase. Such an alignment permits the identification of the glutamic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1 to be substituted with an aspartic acid residue. It will also be appreciated that not all transferases can accept acetoacetyl-CoA as a substrate. As such, those transferases that can accept acetoacetyl-CoA as a substrate are preferred for use in the methods of the disclosure. Optionally, the engineered acetoacetyl-CoA specific hydrolase may be further modified by any methods known in the art including, by random mutagenesis, to increase hydrolase activity.

Exemplary enzymes suitable to accept acetoacetyl-CoA as substrate are set forth in Table 2 and are found among the subfamily of transferases acting at 3-oxoacids (Table 1). These enzymes can be engineered to not consume the equimolar amount of the acceptor acid molecule as co-substrate, but instead perform the hydrolysis of the thioester bound and liberate acetoacetate and free Coenzyme A (HCoA) as products. With the exception of Uniprot No. P37766 (this sequence is a fusion of an alpha and beta subunit), Table 1 lists the beta subunit of a CoA-transferase. CoA-transferases are comprised of an alpha subunit and a beta subunit, and as such, those beta subunits listed in Table 1 must be combined with an alpha subunit in order to produce a catalytically active CoA-transferase. It will be understood that the CoA-transferase beta subunits listed in Table 1 may be combined with any known CoA-transferase alpha subunit that renders the combination of the beta subunit and alpha subunit catalytically active.


TABLE 1
Exemplary 3-oxoacid CoA-transferases.
Gene
Uniprot Entry
Catalytic
Protein Name
Name
Organism
Uniprot
Name
Glu
Acetate CoA-
atoA
Escherichia
P76459
ATOA_ECOLI
E46
transferase subunit
coli (strain
beta
K12)
Acetate CoA-
YdiF
Escherichia
P37766
YDIF_ECOLI
E333
transferase
coli (strain
K12)
Acetate CoA-
atoA
Haemophilus
P44874
ATOA_HAEIN
E46
transferase subunit
influenzae
beta
3-oxoadipate CoA-
catJ
Pseudomonas
Q8VPF2
CATJ_PSESB
E51
transferase subunit
sp.
B
Butyrate-
ctfB
Clostridium
P23673
CTFB_CLOAB
E51
acetoacetate CoA-
acetobutylicum
transferase subunit
B
Glutaconate CoA-
gctB
Acidaminococcus
Q59112
GCTB_ACIFV
E54
transferase subunit
fermentans
B
(strain ATCC
25085/
DSM 20731/
VR4)
3-oxoadipate CoA-
pcaJ
Acinetobacter
Q59091
PCAJ_ACIAD
E50
transferase subunit
catJ
sp. (strain ADP1)
3-oxoadipate CoA-
pcaJ
Pseudomonas
P0A101
PCAJ_PSEPK
E50
transferase subunit
putida (strain
KT2440)
3-oxoadipate CoA-
pcaJ
Pseudomonas
P0A102
PCAJ_PSEPU
E50
transferase subunit
putida
(Arthrobacter
siderocapsulatus)
Probable succinyl-
scoB
Bacillussubtilis
P42316
SCOB_BACSU
E47
CoA:3-ketoacid
(strain 168)
coenzyme A
Succinyl-CoA:3-
scoB
Helicobacter
Q9ZLE4
SCOB_HELPJ
E43
ketoacid
pylori (strain
coenzyme A
J99)
transferase
(Campylobacter
pylori J99)
Succinyl-CoA:3-
scoB
Helicobacter
P56007
SCOB_HELPY
E43
ketoacid
pylori (strain
coenzyme A
ATCC 700392/
transferase
26695)
(Campylobacter
pylori)
Probable succinyl-
scoB
Mycobacterium
P63651
SCOB_MYCBO
E50
CoA:3-ketoacid
bovis (strain
coenzyme A
ATCC BAA-935/
transferase
AF2122/97)
Probable succinyl-
scoB
Mycobacterium
P63650
SCOB_MYCTU
E50
CoA:3-ketoacid
tuberculosis
coenzyme A
transferase
Succinyl-CoA:3-
IpsJ
Xanthomonas
B0RVK3
SCOB_XANCB
E47
ketoacid
campestris pv.
coenzyme A
campestris
transferase
(strain B100)
Succinyl-CoA:3-
IpsJ
Xanthomonas
P0C718
SCOB_XANCP
E47
ketoacid
campestris pv.
coenzyme A
campestris
transferase
(strain ATCC
33913/NCPPB
528/LMG 568)
Putative CoA-
Rv3552
Mycobacterium
P63652
Y3552_MYCTU
E52
transferase subunit
tuberculosis
beta Rv3552
Putative CoA-
Mb3582
Mycobacterium
P63653
Y3582_MYCBO
E52
transferase subunit
bovis (strain
beta Mb3582
ATCC BAA-935/
AF2122/97)
Probable
yodR
Bacillussubtilis
O34466
YODR_BACSU
E50
coenzyme A
(strain 168)
transferase subunit
beta


TABLE 2
Exemplary 3-oxoacid CoA-transferases able to accept acetoacetyl-CoA as
substrate.
Gene
Protein Names
Names
Organism
Uniprot
Entry Name
Acetate CoA-
atoA
Escherichiacoli
P76459
ATOA_ECOLI
transferase
(strain K12)
subunit beta
Acetate CoA-
YdiF
Escherichiacoli
P37766
YDIF_ECOLI
transferase
(strain K12)
Butyrate-acetoacetate
ctfB
Clostridium
P23673
CTFB_CLOAB
CoA-transferase
acetobutylicum
subunit B
Probable succinyl-
scoB
Bacillussubtilis
P42316
SCOB_BACSU
CoA:3-ketoacid
(strain 168)
coenzyme A
transferase

Modification of Microorganism

A microorganism may be modified (e.g., genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to one or more products. Such microorganism may comprise a polynucleotide coding for an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity (e.g., an enzyme that comprises i) an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity and ii) a substitution of a glutamic acid residue to an aspartic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1).

Pathways that utilize an engineered enzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoA specific hydrolase activity are shown below. Such pathways are merely exemplary and represent a few of the ways in which the engineered enzyme disclosed herein may be exploited to catalyze the conversion of a fermentable carbon source to one or more desired end-products.

In some embodiments, a microorganism may be modified (e.g., genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to intermediates in a pathway for the co-production of 1-propanol and 2-propanol. Such enzymes may include any of those enzymes as set forth in FIG. 4 or 5. For example, the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of dihydroxyacetone phosphate or pyruvate to 1-propanol.

In some embodiments, the microorganism may comprise one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 1-propanol and 2-propanol from a fermentable carbon source under anaerobic conditions.

In some embodiments, the microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 2-propanol including, for example, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetate to acetone, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetone to 2-propanol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 3.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol including, for example: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 4.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1-propanol including, for example, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 5.

A modified microorganism as provided herein may comprise:

    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to pyruvate,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to cytosolic acetyl-CoA,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetoacetyl-CoA to AcAcetate,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of AcAcetate to acetone,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetone to 2-propanol,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of methylglyoxal to lactaldehyde,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of methylglyoxal to hydroxyacetone,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of hydroxyacetone to 1,2-propanediol,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactaldehyde to 1,2-propanediol,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to propionaldehyde, and/or
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of propionaldehyde to 1-propanol.

      In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6). In some embodiments, the modified microorganism is capable of growth on a C6 carbon source under anaerobic conditions. In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6) and is capable of growth on a C6 carbon source under anaerobic conditions.

A modified microorganism as provided herein may comprise:

    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to lactate,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to lactoyl-CoA,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactoyl-CoA to lactaldehyde,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid;
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactaldehyde to 1,2-propanediol,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to propionaldehyde,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of propionaldehyde to 1-propanol,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to acetyl-CoA,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetoacetyl-CoA to AcAcetate,
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of AcAcetate to acetone, and/or
    • one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetone to 2-propanol.

      In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). In some embodiments, the modified microorganism is capable of growth on a C6 carbon source under anaerobic conditions. In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable of growth on a C6 carbon source under anaerobic conditions.

Exemplary enzymes that convert a fermentable carbon source such as glucose to 1-propanol (Pathways B and C) and/or 2-propanol (Pathway A) including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Tables 3 to 5 below. The enzyme reference identifier listed in Tables 3 to 5 correlates with the enzyme numbering used in FIGS. 4 and 5, which schematically represents the enzymatic conversion of a fermentable carbon source such as glucose to dihydroxyacetone phosphate or lactate and pyruvate. Dihydroxyacetone phosphate or lactate and pyruvate may be further converted to 1-propanol and/or 2-propanol, using any combination of those enzymes provided in Tables 3 to 5 above including, all of those enzymes as provided in Table 3 to 5 below.


TABLE 3
Pathway A (2-propanol from pyruvate)
Enzyme
EC
No.
Enzyme name
Number
Reaction
A1.
Formate-C acetyltransferase
2.3.1.54
Pyruvate + CoA →
Formate-C acetyltransferase
1.97.1.4
Acetyl-CoA + formate
activating enzyme
A2.
Pyruvate dehydrogenase
1.2.4.1
Pyruvate + CoA +
2.3.1.12
NAD+→ Acetyl-CoA +
1.8.1.4
CO2 + NADH
B.
Thiolase
2.3.1.9
2 acetyl-Coa →
acetoacetyl-CoA + CoA
C.
Acetoacetyl-CoA
2.8.3.8
acetoacetyl-Coa +
acetyltransferase
acetate → acetoacetate +
(engineered as described
acetyl-CoA
herein)
D.
Acetatoacetate
4.1.1.4
acetoacetate →
decarboxylase
acetone + CO2
E.
Secondary alcohol
1.1.1.2
acetone + NAD(P)H→
dehydrogenase
2-propanol + NAD(P)+


TABLE 4
Pathway B (1-propanol from Dihydroxyacetone phosphate
Enzyme
EC
No.
Enzyme name
Number
Reaction
F1.
methylglyoxal
4.2.3.3
dihydroxyacetone phosphate →
synthase
methylglyoxal
F2.
methylglyoxal
4.2.3.3
dihydroxyacetone phosphate →
synthase, phosphate
methylglyoxal
insensitive
G.
Methylglyoxal
1.1.1.-
Methylglyoxal → lactaldehyde
reductase
H.
Methylglyoxal
1.1.1.78
methylglyoxal →
reductase
hydroxyacetone
I.
methylglyoxal
1.1.1.-
Hydroxyacetone + NAD(P)H +
reductase
H+→ 1,2-propanediol +
[multifunctional]
NAD(P)+
J.
methylglyoxal
1.1.1.-
Lactaldehyde + NAD(P)H +
reductase
H+→ 1,2-propanediol +
[multifunctional]
NAD(P)+
K.
1,2 propanediol
4.2.1.30
R/S 1,2 propanediol →
dehydratase
proprionaldehyde
L.
1-propanol
1.1.1.-
proprionaldehyde + NADH →
dehydrogenase
propanol + NAD+


TABLE 5
Pathway C (1-propanol from lactate)
Enzyme
EC
No.
Enzyme name
Number
Reaction
M1.
D-Lactate
1.1.1.28
Pyruvate + NAD(P)H + H+
dehydrogenase
D-Lactate + NAD(P)+
M2.
L-Lactate
1.1.1.27
Pyruvate + NAD(P)H + H+
dehydrogenase
L-Lactate + NAD(P)+
N.
Propionate CoA-
2.8.3.1
Lactate + Acetyl-CoA → lactoyl-
transferase*
CoA + _acetic acid
O.
Lactoyl-CoA
2.3.3.-
Lactate + CoA + ATP → lactoyl-
synthase
CoA + AMP
P.
1,2-propanediol
1.2.1.-
Lactoyl-CoA + NAD(P)H + H+
oxidoreductase
Lactaldehyde + NAD(P)+
Q.
Lactaldehyde
1.1.1.77
L-Lactaldehyde + NAD(P)H +
reductase
H+→ L-1,2-propanediol +
NAD(P)+
J.
methylglyoxal
1.1.1.-
Lactaldehyde + NAD(P)H +
reductase
H+→ 1,2-propanediol +
[multifunctional]
NAD(P)+
K.
1,2 propanediol
4.2.1.28
R/S 1,2 propanediol →
dehydratase
propionaldehyde
L.
1-propanol
1.1.1.-
Propionaldehyde → 1-propanol
dehydrogenase
*enzyme with homologous function but altered substrate specificity is required/preferred

The microorganism may be an archea, bacteria, or eukaryote. In some embodiments, the bacteria is a Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus including, for example, Pelobacter propionicus, Clostridium propionicum, Clostridium acetobutylicum, Lactobacillus, Propionibacterium acidipropionici or Propionibacterium freudenreichii. In some embodiments, the eukaryote is a yeast, filamentous fungi, protozoa, or algae. In some embodiments, the yeast is Saccharomyces cerevisiae, Kluyveromyces lactis or Pichia pastoris.

In some embodiments, the microorganism is additionally modified to comprise one or more tolerance mechanisms including, for example, tolerance to a produced molecule (i.e., methylglyoxal, 1-propanol, 2-propanol, or butadiene), and/or organic solvents. A microorganism modified to comprise such a tolerance mechanism may provide a means to increase titers of fermentations and/or may control contamination in an industrial scale process.

The present disclosure also provides microorganisms (e.g., S. cerevisiae) for the co-production of 2-propanol and 1-propanol and/or 1,2-propanediol. Microorganisms may be modified so that they may co-produce 2-propanol and 1-propanol and/or 1,2-propanediol. In an embodiment, a microorganism may have its native ethanol production reduced or eliminated (i.e., shut off). In an embodiment, to eliminate ethanol production in the microorganism the activity of pyruvate decarboxylase (i.e., the enzyme which decarboxylates pyruvate and in the process makes acetaldehyde and CO2) may be disrupted including, for example, knocked-out. Pyruvate decarboxylase comes in three isoforms in yeast and its activity can be mostly knocked out by deleting the genes PDC1, PDC5, and PDC6. Without wishing to be bound by a theory of the invention, the elimination of the pyruvate decarboxylase activity in the cell's cytoplasm renders the yeast cell unable to grow under anaerobic conditions due to two factors: (1) the lack of an alternative route for cytoplasmic acetyl-CoA production, due to the lack of acetaldehyde that would be converted to acetate and acetyl-coA; and (2) a redox imbalance due to excess NADH because the NADH is no longer oxidized in the conversion of acetaldehyde to ethanol. Thus, it is necessary to also alter the ability of the microorgansim to import glucose by truncating a transcription factor of the glucose importer called MTH1. This truncation then restores the ability of the ΔPDC1,5,6 mutant microorganism to survive on C6 sugars. In an embodiment, one or more polynucleotides coding for a bacterial pyruvate formate lyase or cytosolic pyruvate dehydrogenase complex may be inserted into the microorganism to convert pyruvate into Acetyl CoA in the cytosol. In an embodiment, the microorganism may be modified to comprise one or more polynucleotides that code for enzymes in a pathway for the coproduction of 2-propanol and 1-propanol and/or 1,2-propanediol. In a further embodiment, the microorganism may be modified to comprise an acetoacetylCoA hydrolase. Such an acetoacetylCoA hydrolase may be engineered from an acetoacetylCoA:acetate transferase by making a single Glu-Asp mutation in the acetoacetylCoA:acetate transferase. In an additional embodiment, a microorganism may be modified to comprise one or more polynucleotides coding for a B12-independent dehydratase from the organism Roseburia inuvolurans to convert 1,2-propanediol to propanaldehyde. Microorganisms that comprise one or more of the modifications set forth above are termed a non-naturally occurring microorganism or a modified microorganism.

WO2004099425 discloses the overproduction of pyruvate in S. cerevisiae by knocking out pyruvate decarboxylase activity and a directed evolution process that allowed this triple mutant to grow on glucose due to a truncation of the MTH1 transcription factor. However, the scope stopped at the overproduction of pyruvate in aerobic fermentation systems. The use of oxygen, in this context, was essential as there is a huge buildup of NADH in the cell due to the fact that NADH is no longer oxidized in the conversion of acetaldehyde to ethanol.

The present disclosure also provides modified microorganisms that comprise: a disruption of one or more enzymes that decarboxylate pyruvate and/or a disruption of one or more transcription factors of one or more enzymes that decarboxylate pyruvate; a genetic modification that substantially decreases glucose import into the microorganism; one or more polynucleotides encoding an acetoacetyl-CoA specific hydrolase as disclosed herein, one or more polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of cytosolic acetyl-CoA to 2-propanol; and one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol.

The present disclosure further comprises a pyruvate overproducing cell able to produce cytosolic Acetyl-CoA inserting for example, bacterial pyruvate formate lyase or cytosolic pyruvate dehydrogenase complex to convert pyruvate into Acetyl-CoA in the cytosol of the eukaryote cell. The insertion of pyruvate formate lyase in to a PDC-negative yeast strain was disclosed by Waks and Silver in Engineering a Synthetic Dual-Organism System for Hydrogen Production (Applied and Environmental Microbiology, vol. 75, n. 7, 2009, p. 1867-1875) without success in anaerobic growth or metabolism. Furthermore, the present disclosure further comprises a pyruvate overproducing cell able to produce cytosolic Acetyl-CoA and to grow under anaerobic conditions by providing a temporary redox sink that allows reoxidation of NADH by introducing a gene coding for a bacterial soluble NAD(P)+ transhydrogenase (Si-specific) (udhA gene from E. coli, E.C. number 1.6.1.1.) that catalyzes the interconversion of NADP++NADH=NADPH+NAD+. The concomitant expression of the PFL and udhA enzymes to restore anaerobic growth to the PDC-null yeast strain expressing the truncated MTH1 constitutes the first report of anaerobic growth of a PDC-null yeast strain and serves as a new eukaryotic chassis for the production of commodity chemicals.

In some embodiments, the disclosure contemplates the modification (e.g., engineering) of one or more of the enzymes provided herein. Such modification may be performed to redesign the substrate specificity of the enzyme and/or to modify (e.g., reduce) its activity against others substrates in order to increase its selectivity for a given substrate. Additionally or alternatively, one or more enzymes as provided herein may be engineered to alter (e.g., enhance including, for example, increase its catalytic activity or its substrate specificity) one or more of its properties, including acceptance of different cofactors such as NADH instead of NADPH.

In some embodiments, sequence alignment and comparative modeling of proteins may be used to alter one or more of the enzymes disclosed herein. Homology modeling or comparative modeling refers to building an atomic-resolution model of the desired protein from its primary amino acid sequence and an experimental three-dimensional structure of a similar protein. This model may allow for the enzyme substrate binding site to be defined, and the identification of specific amino acid positions that may be replaced to other natural amino acid in order to redesign its substrate specificity.

Variants or sequences having substantial identity or homology with the polynucleotides encoding enzymes as disclosed herein may be utilized in the practice of the disclosure. Such sequences can be referred to as variants or modified sequences. That is, a polynucleotide sequence may be modified yet still retain the ability to encode a polypeptide exhibiting the desired activity. Such variants or modified sequences are thus equivalents in the sense that they retain their intended function. Generally, the variant or modified sequence may comprise at least about 40%-60%, preferably about 60%-80%, more preferably about 80%-90%, and even more preferably about 90%-95% sequence identity with the native sequence.

In some embodiments, a microorganism may be modified to express including, for example, overexpress, one or more enzymes as provided herein. The microorganism may be modified by genetic engineering techniques (i.e., recombinant technology), classical microbiological techniques, or a combination of such techniques and can also include naturally occurring genetic variants to produce a genetically modified microorganism. Some of such techniques are generally disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press; and Selifonova et al. (2001) Appl. Environ. Microbiol. 67(8):3645).

A genetically modified microorganism may include a microorganism in which a polynucleotide has been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (e.g., over-expression) of one or more enzymes as provided herein within the microorganism. Genetic modifications which result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. Addition of cloned genes to increase gene expression can include maintaining the cloned gene(s) on replicating plasmids or integrating the cloned gene(s) into the genome of the production organism. Furthermore, increasing the expression of desired cloned genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements.

Where desired, the expression of one or more of the enzymes provided herein are under the control of a regulatory sequence that controls directly or indirectly the expression of the enzyme in a time-dependent fashion during a fermentation reaction.

In some embodiments, a microorganism is transformed or transfected with a genetic vehicle such as, an expression vector comprising an exogenous polynucleotide sequence coding for the enzymes provided herein.

Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and may preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

The vectors can be constructed using standard methods (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N. Y. 1989; and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).

The manipulation of polynucleotides of the present disclosure including polynucleotides coding for one or more of the enzymes disclosed herein is typically carried out in recombinant vectors. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed. A vector of use according to the disclosure may be selected to accommodate a protein coding sequence of a desired size. A suitable host cell is transformed with the vector after in vitro cloning manipulations. Host cells may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells. Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene. If given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide repertoire member according to the disclosure.

Vectors, including cloning and expression vectors, may contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. For example, the sequence may be one that enables the vector to replicate independently of the host chromosomal DNA and may include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.

A cloning or expression vector may contain a selection gene also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

The replication of vectors may be performed in E. coli (e.g., strain TB1 or TG1, DH5α, DH10β, JM110). An E. coli-selectable marker, for example, the β-lactamase gene that confers resistance to the antibiotic ampicillin, may be of use. These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.

Expression vectors may contain a promoter that is recognized by the host organism. The promoter may be operably linked to a coding sequence of interest. Such a promoter may be inducible or constitutive. Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.

Promoters suitable for use with prokaryotic hosts may include, for example, the α-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Moreover, host constitutive or inducible promoters may be used. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.

Viral promoters obtained from the genomes of viruses include promoters from polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2 or 5), herpes simplex virus (thymidine kinase promoter), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (e.g., MoMLV, or RSV LTR), Hepatitis B virus, Myeloproliferative sarcoma virus promoter (MPSV), VISNA, and Simian Virus 40 (SV40). Heterologous mammalian promoters include, e.g., the actin promoter, immunoglobulin promoter, heat-shock protein promoters.

The early and late promoters of the SV40 virus are conveniently obtained as a restriction fragment that also contains the SV40 viral origin of replication (see, e.g., Fiers et al., Nature, 273:113 (1978); Mulligan and Berg, Science, 209:1422-1427 (1980); and Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981)). The immediate early promoter of the human cytomegalovirus (CMV) is conveniently obtained as a Hind III E restriction fragment (see, e.g., Greenaway et al., Gene, 18:355-360 (1982)). A broad host range promoter, such as the SV40 early promoter or the Rous sarcoma virus LTR, is suitable for use in the present expression vectors.

Generally, a strong promoter may be employed to provide for high level transcription and expression of the desired product. Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV or CMV IE). In an embodiment, the promoter is a SV40 or a CMV early promoter.

The promoters employed may be constitutive or regulatable, e.g., inducible. Exemplary inducible promoters include jun, fos and metallothionein and heat shock promoters. One or both promoters of the transcription units can be an inducible promoter. In an embodiment, the GFP is expressed from a constitutive promoter while an inducible promoter drives transcription of the gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.

The transcriptional regulatory region in higher eukaryotes may comprise an enhancer sequence. Many enhancer sequences from mammalian genes are known e.g., from globin, elastase, albumin, α-fetoprotein and insulin genes. A suitable enhancer is an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the enhancer of the cytomegalovirus immediate early promoter (Boshart et al. Cell 41:521 (1985)), the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers (see also, e.g., Yaniv, Nature, 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters). The enhancer sequences may be introduced into the vector at a position 5′ or 3′ to the gene of interest, but is preferably located at a site 5′ to the promoter.

Yeast and mammalian expression vectors may contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Therefore, the vector may have other components such as an origin of replication (e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected host cells), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional eukaryotic selectable gene(s) may be incorporated. Generally, in cloning vectors the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known, e.g., the ColE1 origin of replication in bacteria. Various viral origins (e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, a eukaryotic replicon is not needed for expression in mammalian cells unless extrachromosomal (episomal) replication is intended (e.g., the SV40 origin may typically be used only because it contains the early promoter).

To facilitate insertion and expression of different genes coding for the enzymes as disclosed herein from the constructs and expression vectors, the constructs may be designed with at least one cloning site for insertion of any gene coding for any enzyme disclosed herein. The cloning site may be a multiple cloning site, e.g., containing multiple restriction sites.

The plasmids may be propagated in bacterial host cells to prepare DNA stocks for subcloning steps or for introduction into eukaryotic host cells. Transfection of eukaryotic host cells can be any performed by any method well known in the art. Transfection methods include lipofection, electroporation, calcium phosphate co-precipitation, rubidium chloride or polycation mediated transfection, protoplast fusion and microinjection. Preferably, the transfection is a stable transfection. The transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type, is favored. Suitable methods can be determined by routine procedures. For stable transfectants, the constructs are integrated so as to be stably maintained within the host chromosome.

Vectors may be introduced to selected host cells by any of a number of suitable methods known to those skilled in the art. For example, vector constructs may be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors. For example, standard calcium-chloride-mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation and conjugation may also be used (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods may be used (e.g., Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells may be isolated on selective media appropriate to the selectable marker used. Alternatively, or in addition, plates or filters lifted from plates may be scanned for GFP fluorescence to identify transformed clones.

For the introduction of vectors comprising differentially expressed sequences to mammalian cells, the method used may depend upon the form of the vector. Plasmid vectors may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, lipofection reagents such as LIPOFECTAMINE™ (Life Technologies) or LIPOTAXI™ (Stratagene) kits are available. Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

The host cell may be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation. Immortalized host cell cultures amenable to transfection and in vitro cell culture and of the kind typically employed in genetic engineering are preferred. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); human embryonic kidney line (293 or 293 derivatives adapted for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); DHFR-Chinese hamster ovary cells (ATCC CRL-9096); dp12.CHO cells, a derivative of CHO/DHFR-(EP 307,247 published 15 Mar. 1989); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); PEER human acute lymphoblastic cell line (Ravid et al. Int. J. Cancer 25:705-710 (1980)); MRC 5 cells; FS4 cells; human hepatoma line (Hep G2), human HT1080 cells, KB cells, JW-2 cells, Detroit 6 cells, NIH-3T3 cells, hybridoma and myeloma cells. Embryonic cells used for generating transgenic animals are also suitable (e.g., zygotes and embryonic stem cells).

Suitable host cells for cloning or expressing polynucleotides (e.g., DNA) in vectors may include, for example, prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), E. coli JM110 (ATCC 47,013) and E. coli W3110 (ATCC 27,325) are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast may be suitable cloning or expression hosts for vectors comprising polynucleotides coding for one or more enzymes. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

When the enzyme is glycosylated, suitable host cells for expression may be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silk moth) have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as host cells.

Examples of useful mammalian host cells are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-described expression or cloning vectors for production of one or more enzymes as disclosed herein or with polynucleotides coding for one or more enzymes as disclosed herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Host cells containing desired nucleic acid sequences coding for the disclosed enzymes may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44, (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adeNOSine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Any of the intermediates produced in any of the enzymatic pathways disclosed herein may be an intermediate in the classical sense of the word in that they may be enzymatically converted to another intermediate or an end product. Alternatively, the intermediates themselves may be considered an end product.

Polynucleotides and Encoded Enzymes

Any known polynucleotide (e.g., gene) that codes for an enzyme or variant thereof that is capable of catalyzing an enzymatic conversion including, for example, an enzyme as set forth in any one of Tables 3-5 or FIGS. 4-5, is contemplated for use by the present disclosure. Such polynucleotides may be modified (e.g., genetically engineered) to modulate (e.g., increase or decrease) the substrate specificity of an encoded enzyme, or the polynucleotides may be modified to change the substrate specificity of the encoded enzyme (e.g., a polynucleotide that codes for an enzyme with specificity for a substrate may be modified such that the enzyme has specificity for an alternative substrate). Preferred microorganisms may comprise polynucleotides coding for one or more of the enzymes as set forth in Tables 3-5 and FIGS. 4-5.

Enzymes for catalyzing the conversions set forth in pathways A, B, and C of Tables 3-5 and FIGS. 4-5 are categorized in Table 4 below.


TABLE 4
Exemplary Gene Identifier (GI) numbers
Uniprot
SEQ
Pathways
FIGS.
Enzyme No.
EC No.
Enzyme candidate
Gene
ID (aa)
ID NO.
A
4, 5
A
2.3.1.54/
Formate-C
PFLB
P75793
7
1.97.1.4
acetyltransferase
A
4, 5
A
2.3.1.54/
Formate-C
PFLA
C4ZXZ6
8
1.97.1.4
acetyltransferase
(activating enzyme)
A
4, 5
A
2.3.1.54/
Formate-C
PFLB
K9LI23
9
1.97.1.4
acetyltransferase
A
4, 5
A
2.3.1.54/
Formate-C
PFLA
Q6RFH6
10
1.97.1.4
acetyltransferase
(activating enzyme)
A
4, 5
A
1.2.4.1/
Pyruvate
pda1
P16387
11
2.3.1.12/
dehydrogenase complex
1.8.1.4
A
4, 5
A
1.2.4.1/
Pyruvate
pdb1
P32473
12
2.3.1.12/
dehydrogenase complex
1.8.1.4
A
4, 5
A
1.2.4.1/
Pyruvate
lat1
P12695
13
2.3.1.12/
dehydrogenase complex
1.8.1.4
A
4, 5
A
1.2.4.1/
Pyruvate
lpd1
P09624
14
2.3.1.12/
dehydrogenase complex
1.8.1.4
A
4, 5
A
1.2.4.1/
Pyruvate
pdx1
P16451
15
2.3.1.12/
dehydrogenase complex
1.8.1.4
A
4, 5
A
1.2.4.1/
Pyruvate
pdhA
F2MRX7
16
2.3.1.12/
dehydrogenase complex
1.8.1.4
(E1 aplha)
A
4, 5
B
3.1.2.—
Acetoacetyl-CoA
SEQ ID
hydrolase
NO: 2,
4, or 6
A
4, 5
D
4.1.1.4
acetoacetate
adc
P23670
17
decarboxylase
A
4, 5
D
4.1.1.4
acetoacetate
adc
A6M020
18
decarboxylase
A
4, 5
E
1.1.1.2
secondary alcohol
adh
P25984
19
dehydrogenase
B
4
F
4.2.3.3
methylglyoxal synthase
mgsA
P42980
20
B
4
F
4.2.3.3
methylglyoxal synthase
mgsA
P0A731
21
B
4
F
4.2.3.3
methylglyoxal synthase
mgsA*
P0A731
22
B
4
G
1.1.1.—
methylglyoxal reductase,
ydjg
P77256
23
multifunctional
B
4
H
1.1.1.78
methylglyoxal reductase
ypr1
C7GMG9
24
B
4
I
1.1.1.304
methylglyoxal reductase,
budC
Q48436
25
multifunctional
B, C
4, 5
J
1.1.1.77
lactaldehyde reductase
fucO
P0A9S1
26
B, C
4, 5
J
1.1.1.—
methylglyoxal reductase
yafB
P30863
27
[multifunctional]
B, C
4, 5
K
4.2.1.30
glycerol dehydratase
dhaB1
Q8GEZ8
28
B, C
4, 5
K
4.2.1.30
glycerol dehydratase
dhaB2
Q8GEZ7
29
activator
B, C
4, 5
K
4.2.1.30
diol dehydratase
b1
Q1A666
30
B, C
4, 5
K
4.2.1.30
diol dehydratase
b2
Q1A665
31
activator
B, C
4, 5
L
1.1.1.1
alcohol dehydrogenase
adh
C6PZV5
32
C
5
M
1.1.1.28
D-Lactate
ldhA
P52643
33
dehydrogenase
C
5
M
1.1.1.27
L-Lactate
ldhL2
P59390
34
dehydrogenase
C
5
M
1.1.1.27
L-lactate
ldh2
P19858
35
dehydrogenase
C
5
N
2.8.3.1
propionate CoA-
pct
Q9L3F7
36
transferase*
C
5
O
2.3.3.—
Lactoyl-CoA Synthase
ACS1
Q01574
37
C
5
P
1.2.1.—
CoA-dependent
pduP
Q9XDN1
38
propionaldehyde
dehydrogenase*
C
5
Q
1.1.1.77
L-1,2-propanediol
fucO
P0A9S1
39
oxidoreductase

Methods for the Co-Production of 1-Propanol and 2-Propanol

1-propanol and 2-propanol may be produced by contacting any of the genetically modified microorganisms provided herein with a fermentable carbon source. Such methods may preferably comprise contacting a fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to any of the intermediates provided in FIGS. 4-5 (Tables 3-5) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates provided in FIGS. 4-5 (tables 3-5) to 1-propanol and 2-propanol in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to the one or more intermediates provided in FIGS. 4-5 (tables 3-5) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates provided in FIGS. 4-5 (tables 3-5) to 1-propanol and 2-propanol.

The metabolic pathways that lead to the production of industrially important compounds involve oxidation-reduction (redox) reactions. For example, during fermentation, glucose is oxidized in a series of enzymatic reactions into smaller molecules with the concomitant release of energy. The electrons released are transferred from one reaction to another through universal electron carriers, such Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)), which act as cofactors for oxidoreductase enzymes. In microbial catabolism, glucose is oxidized by enzymes using the oxidized form of the cofactors (NAD(P)+ and/or NAD+) as cofactor thus generating reducing equivalents in the form of the reduced cofactor (NAD(P)H and NADH). In order for fermentation to continue, redox-balanced metabolism is required, i.e., the cofactors must be regenerated by the reduction of microbial cell metabolic compounds.

Microorganism-catalyzed fermentation for the production of natural products is a widely known application of biocatalysis. Industrial microorganisms can affect multistep conversions of renewable feedstocks to high value chemical products in a single reactor. Products of microorganism-catalyzed fermentation processes range from chemicals such as ethanol, lactic acid, amino acids and vitamins, to high value small molecule pharmaceuticals, protein pharmaceuticals, and industrial enzymes. In many of these processes, the biocatalysts are whole-cell microorganisms, including microorganisms that have been genetically modified to express heterologous genes.

Some key parameters for efficient microorganism-catalyzed fermentation processes include the ability to grow microorganisms to a greater cell density, increased yield of desired products, increased amount of volumetric productivity, removal of unwanted co-metabolites, improved utilization of inexpensive carbon and nitrogen sources, adaptation to varying fermenter conditions, increased production of a primary metabolite, increased production of a secondary metabolite, increased tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions and increased tolerance to high or low temperatures. Inefficiencies in any of these parameters can result in high manufacturing costs, inability to capture or maintain market share, and/or failure to bring fermented end-products to market.

The methods and compositions of the present disclosure can be adapted to conventional fermentation bioreactors (e.g., batch, fed-batch, cell recycle, and continuous fermentation).

In some embodiments, a microorganism (e.g., a genetically modified microorganism) as provided herein is cultivated in liquid fermentation media (i.e., a submerged culture) which leads to excretion of the fermented product(s) into the fermentation media. In one embodiment, the fermented end product(s) can be isolated from the fermentation media using any suitable method known in the art.

In some embodiments, formation of the fermented product occurs during an initial, fast growth period of the microorganism. In one embodiment, formation of the fermented product occurs during a second period in which the culture is maintained in a slow-growing or non-growing state. In one embodiment, formation of the fermented product occurs during more than one growth period of the microorganism. In such embodiments, the amount of fermented product formed per unit of time is generally a function of the metabolic activity of the microorganism, the physiological culture conditions (e.g., pH, temperature, medium composition), and the amount of microorganisms present in the fermentation process.

In some embodiments, the fermentation product is recovered from the periplasm or culture medium as a secreted metabolite. In one embodiment, the fermentation product is extracted from the microorganism, for example when the microorganism lacks a secretory signal corresponding to the fermentation product. In one embodiment, the microorganisms are ruptured and the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions may then be separated if necessary. The fermentation product of interest may then be purified from the remaining supernatant solution or suspension by, for example, distillation, fractionation, chromatography, precipitation, filtration, and the like.

The methods of the present disclosure are preferably preformed under anaerobic conditions. Both the degree of reduction of a product as well as the ATP requirement of its synthesis determines whether a production process is able to proceed aerobically or anaerobically. To produce 1-propanol and 2-propanol or 1-propanol and butadiene via anaerobic microbial conversion, or at least by using a process with reduced oxygen consumption, redox imbalances should be avoided. Several types of metabolic conversion steps involve redox reactions. Such redox reactions involve electron transfer mediated by the participation of redox cofactors such as NADH, NADPH and ferredoxin. Since the amounts of redox cofactors in the cell are limited to permit the continuation of metabolic processes, the cofactors have to be regenerated. In order to avoid such redox imbalances, alternative ways of cofactor regeneration may be engineered, and in some cases additional sources of ATP generation may be provided. Alternatively, oxidation and reduction processes may be separated spatially in bioelectrochemical systems (Rabaey and. Rozendal, 2010, Nature reviews, Microbiology, vol 8: 706-716).

In some embodiment, redox imbalances may be avoided by using substrates (e.g., fermentable carbon sources) that are more oxidized or more reduced. for example, if the utilization of a substrate results in a deficit or surplus of electrons, a requirement for oxygen can be circumvented by using substrates that are more reduced or oxidized, respectively. For example, glycerol which is a major byproduct of biodiesel production is more reduced than sugars, and is therefore more suitable for the synthesis of compounds whose production from sugar results in cofactor oxidation, such as succinic acid. In some embodiments, if the conversion of a substrate to a product results in an electron deficit, co-substrates can be added that function as electron donors (Babel 2009, Eng. Life Sci. 9, 285-290). An important criterion for the anaerobic use of co-substrates is that their redox potential is higher than that of NADH (Geertman et al., 2006, FEMS Yeast Res. 6, 1193-1203). If the conversion of substrate to produce results in an electron surplus, co-substrates can be added that function as electron acceptors.

Methods for the Production of Polypropylene

1-propanol produced via methods disclosed herein may be dehydrated to form propylene, which may then be polymerized to produce polypropylene in a cost-effective manner.

Propylene is a chemical compound that is widely used to synthesize a wide range of petrochemical products. For instance, this olefin is the raw material used for the production of polypropylene, its copolymers and other chemicals such as acrylonitrile, acrylic acid, epichloridrine and acetone. Propylene demand is growing faster than ethylene demand, mainly due to the growth of market demand for polypropylene. Propylene is polymerized to produce thermoplastics resins for innumerous applications such as rigid or flexible packaging materials, blow molding and injection molding.

Propylene is typically obtained in large quantity scales as a byproduct of catalytical or thermal oil cracking, or as a co-product of ethylene production from natural gas. (Propylene, Jamie G. Lacson, CEH Marketing Research Report-2004, Chemical Economics Handbook-SRI International). The use of alternative routes for the production of propylene has been continuously evaluated using a wide range of renewable raw materials (“Green Propylene”, Nexant, January 2009). These routes include, for example, dimerization of ethylene to yield butylene, followed by metathesis with additional ethylene to produce propylene. Another route is biobutanol production by sugar fermentation followed by dehydration and methatesis with ethylene. Some thermal routes are also being evaluated such as gasification of biomass to produce a syngas followed by synthesis of methanol, which may then produce green propylene via methanol-to-olefin technology.

Propylene production by iso-propanol dehydration has been well-described in document EP00498573B1, wherein all examples show propylene selectivity higher than 90% with high conversions. Dehydration of 1-propanol has also been studied in the following articles: “Mechanism and Kinetics of the Acid-Catalyzed Dehydration of 1- and iso-propanol in Hot Compressed Liquid Water” (Antal, M et al., Ind. Eng. Chem. Res. 1998, 37, 3820-3829) and “Fischer-Tropsch Aqueous Phase Refining by Catalytic Alcohol Dehydration” (Nel, R. et al., Ind. Eng. Chem. Res. 2007, 46, 3558-3565). The reported yield is higher than 90%.

EXAMPLES

Example 1: Engineering of Acetoacetyl-CoA Hydrolase

An enzyme having acetoacetyl-CoA transferase activity may be engineered by any method known in the art to produce an acetoacetyl-CoA specific hydrolase.

In an exemplary method, an amino acid sequence of an enzyme having acetoacetyl-CoA transferase activity is obtained. Next, the glutamic acid residue at a position corresponding to amino acid position 51 of SEQ ID NO: 1 in the enzyme is identified by aligning the amino acid sequence of the enzyme with SEQ ID NO: 1. A site in the enzyme corresponding to amino acid position 51 of SEQ ID NO: 1 is then selected for substitution. Such substitution of the identified glutamic acid residue may include substitution of the glutamic acid residue for aspartic acid and may be made by any method known in the art including, for example, site directed mutagenesis. Subsequently, an acetoacetyl-CoA specific hydrolase is obtained having a specific acetoacetyl-CoA hydrolase activity at least 10× higher than its acetyl-CoA hydrolase activity.

Example 2: Modification of Microorganism for Production of 1-Propanol and 2-Propanol

A microorganism such as a bacterium is genetically modified to produce 1-propanol and 2-propanol from a fermentable carbon source including, for example, glucose.

In an exemplary method, a microorganism may be genetically engineered by any methods known in the art to comprise: i.) one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to dihydroxyacetone-phosphate or glyceraldehyde 3-phosphate and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate or glyceraldehyde 3-phosphate to 1-propanol and 2-propanol.

Alternatively, a microorganism that lacks one or more enzymes (e.g., one or more functional enzymes that are catalytically active) for the conversion of a fermentable carbon source to 1-propanol and 2-propanol may be genetically modified to comprise one or more polynucleotides coding for enzymes (e.g., functional enzymes including, for example any enzyme disclosed herein) in a pathway that the microorganism lacks to catalyze a conversion of the fermentable carbon source to 1-propanol and 2-propanol.

Example 3: Fermentation of Glucose by Genetically Modified Microorganism to Produce 1-Propanol and 2-Propanol

A genetically modified microorganism, as produced in Example 1 above, may be used to ferment a carbon source to produce 1-propanol and 2-propanol.

In an exemplary method, a previously-sterilized culture medium comprising a fermentable carbon source (e.g., 9 g/L glucose, 1 g/L KH2PO4, 2 g/L (NH4)2HPO4, 5 mg/L FeSO4.7H2O, 10 mg/L MgSO4.7H2O, 2.5 mg/L MnSO4.H2O, 10 mg/L CaCl2.6H2O, 10 mg/L CoCl2.6H2O, and 10 g/L yeast extract) is charged in a bioreactor.

During fermentation, anaerobic conditions are maintained by, for example, sparging nitrogen through the culture medium. A suitable temperature for fermentation (e.g., about 30° C.) is maintained using any method known in the art. A near physiological pH (e.g., about 6.5) is maintained by, for example, automatic addition of sodium hydroxide. The bioreactor is agitated at, for example, about 50 rpm. Fermentation is allowed to run to completion.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms “a,”“an,”“the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein can be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

<160> NUMBER OF SEQ ID NOS: 58

<210> SEQ ID NO: 1

<211> LENGTH: 221

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Butyrate--acetoacetate CoA-

transferase subunit B [Clostridium acetobutylicum]

<400> SEQENCE: 1

Met Ile Asn Asp Lys Asn Leu Ala Lys Glu Ile Ile Ala Lys Arg Val

1 5 10 15

Ala Arg Glu Leu Lys Asn Gly Gln Leu Val Asn Leu Gly Val Gly Leu

20 25 30

Pro Thr Met Val Ala Asp Tyr Ile Pro Lys Asn Phe Lys Ile Thr Phe

35 40 45

Gln Ser Glu Asn Gly Ile Val Gly Met Gly Ala Ser Pro Lys Ile Asn

50 55 60

Glu Ala Asp Lys Asp Val Val Asn Ala Gly Gly Asp Tyr Thr Thr Val

65 70 75 80

Leu Pro Asp Gly Thr Phe Phe Asp Ser Ser Val Ser Phe Ser Leu Ile

85 90 95

Arg Gly Gly His Val Asp Val Thr Val Leu Gly Ala Leu Gln Val Asp

100 105 110

Glu Lys Gly Asn Ile Ala Asn Trp Ile Val Pro Gly Lys Met Leu Ser

115 120 125

Gly Met Gly Gly Ala Met Asp Leu Val Asn Gly Ala Lys Lys Val Ile

130 135 140

Ile Ala Met Arg His Thr Asn Lys Gly Gln Pro Lys Ile Leu Lys Lys

145 150 155 160

Cys Thr Leu Pro Leu Thr Ala Lys Ser Gln Ala Asn Leu Ile Val Thr

165 170 175

Glu Leu Gly Val Ile Glu Val Ile Asn Asp Gly Leu Leu Leu Thr Glu

180 185 190

Ile Asn Lys Asn Thr Thr Ile Asp Glu Ile Arg Ser Leu Thr Ala Ala

195 200 205

Asp Leu Leu Ile Ser Asn Glu Leu Arg Pro Met Ala Val

210 215 220

<210> SEQ ID NO: 2

<211> LENGTH: 221

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Modified butyrate--acetoacetate

CoA-transferase subunit B [Clostridium acetobutylicum]

<400> SEQENCE: 2

Met Ile Asn Asp Lys Asn Leu Ala Lys Glu Ile Ile Ala Lys Arg Val

1 5 10 15

Ala Arg Glu Leu Lys Asn Gly Gln Leu Val Asn Leu Gly Val Gly Leu

20 25 30

Pro Thr Met Val Ala Asp Tyr Ile Pro Lys Asn Phe Lys Ile Thr Phe

35 40 45

Gln Ser Asp Asn Gly Ile Val Gly Met Gly Ala Ser Pro Lys Ile Asn

50 55 60

Glu Ala Asp Lys Asp Val Val Asn Ala Gly Gly Asp Tyr Thr Thr Val

65 70 75 80

Leu Pro Asp Gly Thr Phe Phe Asp Ser Ser Val Ser Phe Ser Leu Ile

85 90 95

Arg Gly Gly His Val Asp Val Thr Val Leu Gly Ala Leu Gln Val Asp

100 105 110

Glu Lys Gly Asn Ile Ala Asn Trp Ile Val Pro Gly Lys Met Leu Ser

115 120 125

Gly Met Gly Gly Ala Met Asp Leu Val Asn Gly Ala Lys Lys Val Ile

130 135 140

Ile Ala Met Arg His Thr Asn Lys Gly Gln Pro Lys Ile Leu Lys Lys

145 150 155 160

Cys Thr Leu Pro Leu Thr Ala Lys Ser Gln Ala Asn Leu Ile Val Thr

165 170 175

Glu Leu Gly Val Ile Glu Val Ile Asn Asp Gly Leu Leu Leu Thr Glu

180 185 190

Ile Asn Lys Asn Thr Thr Ile Asp Glu Ile Arg Ser Leu Thr Ala Ala

195 200 205

Asp Leu Leu Ile Ser Asn Glu Leu Arg Pro Met Ala Val

210 215 220

<210> SEQ ID NO: 3

<211> LENGTH: 216

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Acetate CoA--transferase subunit

beta [Escherichia coli]

<400> SEQENCE: 3

Met Asp Ala Lys Gln Arg Ile Ala Arg Arg Val Ala Gln Glu Leu Arg

1 5 10 15

Asp Gly Asp Ile Val Asn Leu Gly Ile Gly Leu Pro Thr Met Val Ala

20 25 30

Asn Tyr Leu Pro Glu Gly Ile His Ile Thr Leu Gln Ser Glu Asn Gly

35 40 45

Phe Leu Gly Leu Gly Pro Val Thr Thr Ala His Pro Asp Leu Val Asn

50 55 60

Ala Gly Gly Gln Pro Cys Gly Val Leu Pro Gly Ala Ala Met Phe Asp

65 70 75 80

Ser Ala Met Ser Phe Ala Leu Ile Arg Gly Gly His Ile Asp Ala Cys

85 90 95

Val Leu Gly Gly Leu Gln Val Asp Glu Glu Ala Asn Leu Ala Asn Trp

100 105 110

Val Val Pro Gly Lys Met Val Pro Gly Met Gly Gly Ala Met Asp Leu

115 120 125

Val Thr Gly Ser Arg Lys Val Ile Ile Ala Met Glu His Cys Ala Lys

130 135 140

Asp Gly Ser Ala Lys Ile Leu Arg Arg Cys Thr Met Pro Leu Thr Ala

145 150 155 160

Gln His Ala Val His Met Leu Val Thr Glu Leu Ala Val Phe Arg Phe

165 170 175

Ile Asp Gly Lys Met Trp Leu Thr Glu Ile Ala Asp Gly Cys Asp Leu

180 185 190

Ala Thr Val Arg Ala Lys Thr Glu Ala Arg Phe Glu Val Ala Ala Asp

195 200 205

Leu Asn Thr Gln Arg Gly Asp Leu

210 215

<210> SEQ ID NO: 4

<211> LENGTH: 216

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Modified Acetate CoA--transferase

subunit beta [Escherichia coli]

<400> SEQENCE: 4

Met Asp Ala Lys Gln Arg Ile Ala Arg Arg Val Ala Gln Glu Leu Arg

1 5 10 15

Asp Gly Asp Ile Val Asn Leu Gly Ile Gly Leu Pro Thr Met Val Ala

20 25 30

Asn Tyr Leu Pro Glu Gly Ile His Ile Thr Leu Gln Ser Asp Asn Gly

35 40 45

Phe Leu Gly Leu Gly Pro Val Thr Thr Ala His Pro Asp Leu Val Asn

50 55 60

Ala Gly Gly Gln Pro Cys Gly Val Leu Pro Gly Ala Ala Met Phe Asp

65 70 75 80

Ser Ala Met Ser Phe Ala Leu Ile Arg Gly Gly His Ile Asp Ala Cys

85 90 95

Val Leu Gly Gly Leu Gln Val Asp Glu Glu Ala Asn Leu Ala Asn Trp

100 105 110

Val Val Pro Gly Lys Met Val Pro Gly Met Gly Gly Ala Met Asp Leu

115 120 125

Val Thr Gly Ser Arg Lys Val Ile Ile Ala Met Glu His Cys Ala Lys

130 135 140

Asp Gly Ser Ala Lys Ile Leu Arg Arg Cys Thr Met Pro Leu Thr Ala

145 150 155 160

Gln His Ala Val His Met Leu Val Thr Glu Leu Ala Val Phe Arg Phe

165 170 175

Ile Asp Gly Lys Met Trp Leu Thr Glu Ile Ala Asp Gly Cys Asp Leu

180 185 190

Ala Thr Val Arg Ala Lys Thr Glu Ala Arg Phe Glu Val Ala Ala Asp

195 200 205

Leu Asn Thr Gln Arg Gly Asp Leu

210 215

<210> SEQ ID NO: 5

<211> LENGTH: 531

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Acetate CoA-transferase YdiF

[Escherichia coli]

<400> SEQENCE: 5

Met Lys Pro Val Lys Pro Pro Arg Ile Asn Gly Arg Val Pro Val Leu

1 5 10 15

Ser Ala Gln Glu Ala Val Asn Tyr Ile Pro Asp Glu Ala Thr Leu Cys

20 25 30

Val Leu Gly Ala Gly Gly Gly Ile Leu Glu Ala Thr Thr Leu Ile Thr

35 40 45

Ala Leu Ala Asp Lys Tyr Lys Gln Thr Gln Thr Pro Arg Asn Leu Ser

50 55 60

Ile Ile Ser Pro Thr Gly Leu Gly Asp Arg Ala Asp Arg Gly Ile Ser

65 70 75 80

Pro Leu Ala Gln Glu Gly Leu Val Lys Trp Ala Leu Cys Gly His Trp

85 90 95

Gly Gln Ser Pro Arg Ile Ser Glu Leu Ala Glu Gln Asn Lys Ile Ile

100 105 110

Ala Tyr Asn Tyr Pro Gln Gly Val Leu Thr Gln Thr Leu Arg Ala Ala

115 120 125

Ala Ala His Gln Pro Gly Ile Ile Ser Asp Ile Gly Ile Gly Thr Phe

130 135 140

Val Asp Pro Arg Gln Gln Gly Gly Lys Leu Asn Glu Val Thr Lys Glu

145 150 155 160

Asp Leu Ile Lys Leu Val Glu Phe Asp Asn Lys Glu Tyr Leu Tyr Tyr

165 170 175

Lys Ala Ile Ala Pro Asp Ile Ala Phe Ile Arg Ala Thr Thr Cys Asp

180 185 190

Ser Glu Gly Tyr Ala Thr Phe Glu Asp Glu Val Met Tyr Leu Asp Ala

195 200 205

Leu Val Ile Ala Gln Ala Val His Asn Asn Gly Gly Ile Val Met Met

210 215 220

Gln Val Gln Lys Met Val Lys Lys Ala Thr Leu His Pro Lys Ser Val

225 230 235 240

Arg Ile Pro Gly Tyr Leu Val Asp Ile Val Val Val Asp Pro Asp Gln

245 250 255

Thr Gln Leu Tyr Gly Gly Ala Pro Val Asn Arg Phe Ile Ser Gly Asp

260 265 270

Phe Thr Leu Asp Asp Ser Thr Lys Leu Ser Leu Pro Leu Asn Gln Arg

275 280 285

Lys Leu Val Ala Arg Arg Ala Leu Phe Glu Met Arg Lys Gly Ala Val

290 295 300

Gly Asn Val Gly Val Gly Ile Ala Asp Gly Ile Gly Leu Val Ala Arg

305 310 315 320

Glu Glu Gly Cys Ala Asp Asp Phe Ile Leu Thr Val Glu Thr Gly Pro

325 330 335

Ile Gly Gly Ile Thr Ser Gln Gly Ile Ala Phe Gly Ala Asn Val Asn

340 345 350

Thr Arg Ala Ile Leu Asp Met Thr Ser Gln Phe Asp Phe Tyr His Gly

355 360 365

Gly Gly Leu Asp Val Cys Tyr Leu Ser Phe Ala Glu Val Asp Gln His

370 375 380

Gly Asn Val Gly Val His Lys Phe Asn Gly Lys Ile Met Gly Thr Gly

385 390 395 400

Gly Phe Ile Asp Ile Ser Ala Thr Ser Lys Lys Ile Ile Phe Cys Gly

405 410 415

Thr Leu Thr Ala Gly Ser Leu Lys Thr Glu Ile Thr Asp Gly Lys Leu

420 425 430

Asn Ile Val Gln Glu Gly Arg Val Lys Lys Phe Ile Arg Glu Leu Pro

435 440 445

Glu Ile Thr Phe Ser Gly Lys Ile Ala Leu Glu Arg Gly Leu Asp Val

450 455 460

Arg Tyr Ile Thr Glu Arg Ala Val Phe Thr Leu Lys Glu Asp Gly Leu

465 470 475 480

His Leu Ile Glu Ile Ala Pro Gly Val Asp Leu Gln Lys Asp Ile Leu

485 490 495

Asp Lys Met Asp Phe Thr Pro Val Ile Ser Pro Glu Leu Lys Leu Met

500 505 510

Asp Glu Arg Leu Phe Ile Asp Ala Ala Met Gly Phe Val Leu Pro Glu

515 520 525

Ala Ala His

530

<210> SEQ ID NO: 6

<211> LENGTH: 531

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Modified Acetate CoA-transferase

YdiF [Escherichia coli]

<400> SEQENCE: 6

Met Lys Pro Val Lys Pro Pro Arg Ile Asn Gly Arg Val Pro Val Leu

1 5 10 15

Ser Ala Gln Glu Ala Val Asn Tyr Ile Pro Asp Glu Ala Thr Leu Cys

20 25 30

Val Leu Gly Ala Gly Gly Gly Ile Leu Glu Ala Thr Thr Leu Ile Thr

35 40 45

Ala Leu Ala Asp Lys Tyr Lys Gln Thr Gln Thr Pro Arg Asn Leu Ser

50 55 60

Ile Ile Ser Pro Thr Gly Leu Gly Asp Arg Ala Asp Arg Gly Ile Ser

65 70 75 80

Pro Leu Ala Gln Glu Gly Leu Val Lys Trp Ala Leu Cys Gly His Trp

85 90 95

Gly Gln Ser Pro Arg Ile Ser Glu Leu Ala Glu Gln Asn Lys Ile Ile

100 105 110

Ala Tyr Asn Tyr Pro Gln Gly Val Leu Thr Gln Thr Leu Arg Ala Ala

115 120 125

Ala Ala His Gln Pro Gly Ile Ile Ser Asp Ile Gly Ile Gly Thr Phe

130 135 140

Val Asp Pro Arg Gln Gln Gly Gly Lys Leu Asn Glu Val Thr Lys Glu

145 150 155 160

Asp Leu Ile Lys Leu Val Glu Phe Asp Asn Lys Glu Tyr Leu Tyr Tyr

165 170 175

Lys Ala Ile Ala Pro Asp Ile Ala Phe Ile Arg Ala Thr Thr Cys Asp

180 185 190

Ser Glu Gly Tyr Ala Thr Phe Glu Asp Glu Val Met Tyr Leu Asp Ala

195 200 205

Leu Val Ile Ala Gln Ala Val His Asn Asn Gly Gly Ile Val Met Met

210 215 220

Gln Val Gln Lys Met Val Lys Lys Ala Thr Leu His Pro Lys Ser Val

225 230 235 240

Arg Ile Pro Gly Tyr Leu Val Asp Ile Val Val Val Asp Pro Asp Gln

245 250 255

Thr Gln Leu Tyr Gly Gly Ala Pro Val Asn Arg Phe Ile Ser Gly Asp

260 265 270

Phe Thr Leu Asp Asp Ser Thr Lys Leu Ser Leu Pro Leu Asn Gln Arg

275 280 285

Lys Leu Val Ala Arg Arg Ala Leu Phe Glu Met Arg Lys Gly Ala Val

290 295 300

Gly Asn Val Gly Val Gly Ile Ala Asp Gly Ile Gly Leu Val Ala Arg

305 310 315 320

Glu Glu Gly Cys Ala Asp Asp Phe Ile Leu Thr Val Asp Thr Gly Pro

325 330 335

Ile Gly Gly Ile Thr Ser Gln Gly Ile Ala Phe Gly Ala Asn Val Asn

340 345 350

Thr Arg Ala Ile Leu Asp Met Thr Ser Gln Phe Asp Phe Tyr His Gly

355 360 365

Gly Gly Leu Asp Val Cys Tyr Leu Ser Phe Ala Glu Val Asp Gln His

370 375 380

Gly Asn Val Gly Val His Lys Phe Asn Gly Lys Ile Met Gly Thr Gly

385 390 395 400

Gly Phe Ile Asp Ile Ser Ala Thr Ser Lys Lys Ile Ile Phe Cys Gly

405 410 415

Thr Leu Thr Ala Gly Ser Leu Lys Thr Glu Ile Thr Asp Gly Lys Leu

420 425 430

Asn Ile Val Gln Glu Gly Arg Val Lys Lys Phe Ile Arg Glu Leu Pro

435 440 445

Glu Ile Thr Phe Ser Gly Lys Ile Ala Leu Glu Arg Gly Leu Asp Val

450 455 460

Arg Tyr Ile Thr Glu Arg Ala Val Phe Thr Leu Lys Glu Asp Gly Leu

465 470 475 480

His Leu Ile Glu Ile Ala Pro Gly Val Asp Leu Gln Lys Asp Ile Leu

485 490 495

Asp Lys Met Asp Phe Thr Pro Val Ile Ser Pro Glu Leu Lys Leu Met

500 505 510

Asp Glu Arg Leu Phe Ile Asp Ala Ala Met Gly Phe Val Leu Pro Glu

515 520 525

Ala Ala His

530

<210> SEQ ID NO: 7

<211> LENGTH: 2433

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PFLB

<400> SEQENCE: 7

atgaccacac tgaaactgga cacgctcagc gaccgcatta aagcgcacaa aaatgcgctg 60

gtgcatattg tgaaaccgcc agtctgtacc gagcgcgcgc agcactatac cgagatgtat 120

caacaacatc tcgataagcc gatcccggta cgtcgcgcgc tggcactggc gcatcacctg 180

gcgaatcgca ccatctggat caaacacgat gagttgatca ttggcaacca ggcaagcgaa 240

gttcgcgccg cgccgatctt cccggaatat actgtctcgt ggatcgaaaa agagattgat 300

gatctggcag atcgtcccgg tgctggcttt gcggtgagcg aagagaacaa acgcgttctg 360

catgaagtgt gcccgtggtg gcgcggtcag accgtacagg atcgctgcta cggcatgttt 420

accgatgagc aaaaaggtct gctggcgacc ggaatcatta aagcggaagg caatatgacc 480

tccggcgatg cgcacctggc ggtgaatttc ccgctgctgc tggaaaaagg gcttgatggt 540

ctgcgcgagg aagtagcgga acgtcgctcg cgcatcaacc tgacggtgct ggaagattta 600

cacggtgagc aattcctgaa agcgattgat atcgtgctgg tggcagtcag tgaacacatt 660

gaacgtttcg ctgccctggc gcgtgaaatg gccgcgaccg aaacccgcga aagccgtcgc 720

gatgaactgc tggcgatggc agaaaactgc gatcttatcg cccaccagcc gccgcagact 780

ttctggcagg cgctgcaact gtgttacttc atccagttga ttttgcagat cgaatctaac 840

ggtcactcag tatcgtttgg tcgtatggac cagtatctct acccgtacta tcgccgcgac 900

gttgaactca accagacgct ggatcgcgaa cacgccatcg agatgctgca tagctgctgg 960

ctgaaactgc tggaagtgaa caagatccgc tccggctcac actcaaaagc ctctgcggga 1020

agtccgctgt atcagaacgt cactattggc gggcaaaatc tggttgatgg tcaaccaatg 1080

gacgcggtga atccactctc ttacgcgatc ctcgaatcct gcggtcgcct gcgttccact 1140

cagcctaacc tcagcgtgcg ttaccatgca ggaatgagca acgatttcct cgacgcctgc 1200

gtacaggtga tccgttgcgg cttcgggatg ccggcgttca acaacgacga aatcgtgatc 1260

ccggaattta ttaaactcgg tattgaaccg caggacgctt atgactacgc agcgattggt 1320

tgtatagaaa ccgccgtcgg tggcaaatgg ggctatcgct gtaccggcat gagctttatc 1380

aacttcgccc gcgtgatgct ggcggcgctg gaaggcgggc atgatgccac cagcggcaaa 1440

gtgttcctgc cacaagaaaa agcgttgtcg gcaggtaact tcaacaactt cgatgaagtg 1500

atggacgcgt gggatacgca aatccgttac tacacccgca aatcaatcga aatcgaatat 1560

gtcgtcgaca ccatgctgga agagaacgtg cacgatattc tctgctcggc gctggtggat 1620

gactgtattg agcgagcgaa aagtatcaag caaggcggcg cgaaatatga ctgggtttct 1680

ggcctgcagg tcggcattgc caacctcggc aacagcctgg cggcagtgaa gaaactggtg 1740

tttgaacaag gtgcgattgg tcagcaacag cttgctgccg cactggcaga tgacttcgac 1800

ggcctgactc acgagcagct gcgtcagcgg ctgattaacg gtgcgccgaa gtacggcaac 1860

gacgatgata ctgtcgatac gctgctggct cgcgcttatc agacctatat cgacgaactg 1920

aaacagtacc ataatccgcg ctacggtcgt ggtccggttg gcggcaacta ttacgcgggt 1980

acgtcatcaa tctccgctaa cgtaccgttt ggcgcgcaga ctatggcaac accggacggg 2040

cgtaaagccc acaccccgct ggcagaaggc gcaagcccgg cctccggtac tgaccatctt 2100

ggccctactg cggtcattgg ctcagtgggt aaactgccta cggcagcgat tctcggcggc 2160

gtgttgctca accagaaact gaatccggca acgctggaga acgaatctga caagcagaaa 2220

ctgatgatcc tgctgcgtac cttctttgaa gtgcataaag gctggcatat tcagtacaac 2280

atcgtttccc gcgaaacgct gctggatgcg aaaaaacatc ccgatcagta tcgcgatctg 2340

gtagtgcgtg tcgcgggcta ttccgcgttc ttcaccgcgc tctctccaga cgctcaggac 2400

gatatcatcg cccgtactga acatatgctg taa 2433

<210> SEQ ID NO: 8

<211> LENGTH: 927

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PFLA

<400> SEQENCE: 8

atgcttgaac gaaatagaga ggcaactatg attttcaata ttcagcgcta ctcgacccat 60

gatggccccg gtatccgcac ggtcgtattt cttaaaggct gttcgctggg ctgccgctgg 120

tgtcagaacc cggaaagccg cgcccgcacg caggatctgc tgtatgacgc acgactgtgt 180

ctggaaggct gcgagctgtg cgctaaggcc gcgccggaag tgattgagcg cgcgctgaat 240

ggtttgctta ttcatcggga aaagttaacc ccggagcatc tgacggcgtt aaccgactgc 300

tgtccgacac aggcattaac cgtgtgtggt gaagtgaaaa gcgttgagga gatcatgacg 360

accgttctgc gcgataaacc gttttacgat cgcagcggcg gcggtttaac gctttcgggt 420

ggtgagccct ttatgcagcc ggaaatggcg atggcgctac tgcaagccag ccacgaggca 480

ggcattcata ctgcggtaga aacctgtctg catgtgccgt ggaaatatat cgccccttct 540

ctgccctata tcgatctgtt tcttgccgat ttaaaacacg ttgccgacgc gccgtttaaa 600

cagtggaccg acggtaacgc cgccagagtg ctggataacc tgaaaaaact cgccgcagcg 660

ggcaaaaaaa tcattatccg cgtgccgctg attcagggct ttaatgccga cgaaacctct 720

gtaaaagcca ttaccgattt tgccgccgac gagctgcacg ttggcgaaat tcattttctg 780

ccctaccaca cgctgggcat caacaaatat cacttactta atctgcccta tgacgccccg 840

gaaaaaccgc ttgatgcgcc agaactgctc gactttgccc agcagtatgc ctgccagaaa 900

gggttaaccg cgaccttacg aggataa 927

<210> SEQ ID NO: 9

<211> LENGTH: 2415

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PFLB

<400> SEQENCE: 9

atggaaagtt taactttagt caacaacgct cttgtcaagt cagtttcagt taatgctgtt 60

gctgccacta aggttgctgg tgttagaatc agcaagccat ctcgtgctat tcacactact 120

ccaatgacca ctactagtct taaggttgct aagaaggctg ccttctctca atctaagact 180

tatgctactg ctccatgcat tactaatgat gctgctgcca agagtgaaat cgatgttgaa 240

ggttggatta agaagcacta cactccatat gaaggagatg gttctttcct tgctggtcca 300

actgaaaaga ctaagaagct ttttgccaag gctgaagaat acttagccaa ggaacgtgct 360

aacggtggtt tatacgatgt tgacccacac accccatcta ccattacttc tcacaagcca 420

ggttaccttg acaaagaaaa tgaagttatc tacggttacc aaactgatgt tccacttaag 480

agagccatta agccattcgg tggtgttaat atggtaaaga acgctcttaa ggctgttaac 540

gttccaatgg ataaggaagt tgaacacatt ttcactgatt accgtaagac tcacaacact 600

gctgtattcg atatttactc taaggaaatg agagctggtc gttccaatgc tatcatgacc 660

ggtttaccag atggttatgg tcgtggtcgt attattggtg attaccgtcg tgttgccctt 720

tacggtactg accgtcttat tgcccaaaag caaaaggata aggttgaatt acaaaagaga 780

caaatggatg aaccaactat gaaattaatt ggtgaagttg ctgatcaaat taaggctctt 840

aagcaactta ctcaaatggc caagtcttac ggtattgata ttactaagcc agctaagaac 900

gccagagaag ctactcaatt cgtttacttc ggttacttag gttctatcaa ggaacaagat 960

ggtgctgcta tgtctcttgg tcgtgttgat gccttccttg attgtttctt cgaaaatgat 1020

ttaaagaatg gtgttcttga tgaagcccat gcccaagaaa ttattgataa ccttatctta 1080

aagttacgtt tcgctcgtca cttacgtact ccagaataca acgatttatt cgctggtgat 1140

ccaacctggg ttactatgtc tctcggtggt actggttctg atggtcgtac attagttacc 1200

aagacttcct tccgtgttct taacactctt tacaacttag gtccagctcc agaaccaaac 1260

atcactgtcc tttggaacaa gaaccttcca aagaacttta aggactttgc tactaaggtt 1320

tctattgata cctcttccat tcaatacgaa tctgatgctc ttatgtccgc tagattcggt 1380

gatgactacg gtattgcttg ctgtgtctct gccatgagaa ttggtaagga tatgcaattc 1440

ttcggtgctc gttgtaacct tgctaagctt atgctttacg tcctcaacca tggtaaggat 1500

gaaagaactg gtaagcaagt tggtccagac tttggtccag ttccagatgg tccaattcca 1560

ttcgactgga tgtgggaaac ctatgacaag gctatggact ggattgccaa gctttacgtc 1620

aacaccatga acgttattca cttctgccat gaccaatact gttacgaatc ccttcaaatg 1680

gctcttcatg ataccgatgt ccgtcgtctt atggccttcg gtgttgctgg tctttctgtt 1740

gttgctgatt cattctctgc tattaagtac gccaaggtta ctccaatccg tgatccaaag 1800

accggtttaa ctactgactt taaggttgaa ggtgaattcc caaaattcgg taatgatgat 1860

gaccgtgtcg atttcttcgc tcgtaccgtt actgataagc ttattaccaa gttaagaaaa 1920

actccaactt accgtggtgc cactcacact ctttccattc ttaccattac ctctaatgtc 1980

gtttacggta agaagaccgg ttctactcca gatggtcgta aggctggtca accattcgct 2040

ccaggttgta acccaatgca cggtcgtgaa ttctctggtg ctgttgcttc tctttcttca 2100

gtcgctaagg ttaactacga ctcttgtatg gatggtattt ctaacacctt ctctattgtt 2160

ccaaacacca ttggtaagac cttacaagaa cgtcaaggta acctttccgg tttattagat 2220

ggttacttca gcaagggtgc tcaccatctt aacgttaacg ttcttaagcg tgaaacttta 2280

gaagatgcca tggctcaccc agaaaactat ccaaacctta ctattcgtgt ttctggttat 2340

gctgttaact ttgttaagtt aactccagct caacaaaagg aagtcattgc ccgtaccttc 2400

cacgaaaaga tgtaa 2415

<210> SEQ ID NO: 10

<211> LENGTH: 801

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PFLA

<400> SEQENCE: 10

atgccagcta tcgttgatcc aactactatg gattatatgg aagtcaaggg caatgtccat 60

tcaactgaaa gtttggcttg tcttgaaggt ccaggaaaca gattcctttt atttttaaat 120

ggttgtgctg ctcgttgctt atactgtagt aatccagata cttgggatga aactgttggt 180

actccaatga ccgttggcca acttattaag aagattggaa atcttaaaaa ctactatatc 240

aattctgttg gtggtggtgg tgtcactgtt tctggtggtg aaccattaac tcaatttggt 300

ttcttatctt gtttcttata tgctgtcaag aagcacttaa atcttcatac ctgtgttgaa 360

accactggtc aaggttgtac taaggcttgg aattcagttt tacctcatac tgacttatgc 420

ttagtatgta ttaaacatgc tattccagaa aaatacgaac aaattactcg tactaagaaa 480

ttagatagat gtcttaagtt ccttaaggaa ttagaaaaga gaaacattcc atggtggtgt 540

cgttacgttg ttcttccagg ttacactgat tctaaggaag atattgaagc tttaattgaa 600

ttagttaaga acagtccaac ttgtgaaaga attgaattcc ttccataccc cgaattaggt 660

aaaaacaaat gggaagaatt aggtattgaa tatccattaa agaatattaa acaacttaag 720

aaaagtgaaa ttaaatggat ctgtgatatg gtccgtgaag ctttcaagga ccgtaatatt 780

ccagttactg gtgatactta a 801

<210> SEQ ID NO: 11

<211> LENGTH: 1263

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pda1

<400> SEQENCE: 11

atgcttgctg cttcattcaa acgccaacca tcacaattgg tccgcgggtt aggagctgtt 60

cttcgcactc ccaccaggat aggtcatgtt cgtaccatgg caactttaaa aacaactgat 120

aagaaggccc ctgaggacat cgagggctcg gacacagtgc aaattgagtt gcctgaatct 180

tccttcgagt cgtatatgct agagcctcca gacttgtctt atgagacttc gaaagccacc 240

ttgttacaga tgtataaaga tatggtcatc atcagaagaa tggagatggc ttgtgacgcc 300

ttgtacaagg ccaagaaaat cagaggtttt tgccatctat ctgttggtca ggaggccatt 360

gctgtcggta tcgagaatgc catcacaaaa ttggattcca tcatcacatc ttacagatgt 420

cacggtttca cttttatgag aggtgcctca gtgaaagccg ttctggctga attgatgggt 480

agaagagccg gtgtctctta tggtaagggt ggttccatgc acctttacgc tccaggcttc 540

tatggtggta atggtatcgt gggtgcccag gttcctttag gtgcaggttt agcttttgct 600

caccaataca agaacgagga cgcctgctct ttcactttgt atggtgatgg tgcctctaat 660

caaggtcaag tttttgaatc tttcaacatg gccaaattat ggaatttgcc cgtcgtgttt 720

tgctgtgaga acaacaagta cggtatgggt accgccgctt caagatcctc cgcgatgact 780

gaatatttca agcgtggtca atatattcca ggtttaaaag ttaacggtat ggatattcta 840

gctgtctacc aagcatccaa gtttgctaag gactggtgtc tatccggcaa aggtcctctc 900

gttctagaat atgaaaccta taggtacggt ggccattcta tgtctgatcc cggtactacc 960

tacagaacta gagacgagat tcagcatatg agatccaaga acgatccaat tgctggtctt 1020

aagatgcatt tgattgatct aggtattgcc actgaagctg aagtcaaagc ttacgacaag 1080

tccgctagaa aatacgttga cgaacaagtt gaattagctg atgctgctcc tcctccagaa 1140

gccaaattat ccatcttgtt tgaagacgtc tacgtgaaag gtacagaaac tccaacccta 1200

agaggtagga tccctgaaga tacttgggac ttcaaaaagc aaggttttgc ctctagggat 1260

taa 1263

<210> SEQ ID NO: 12

<211> LENGTH: 1101

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pdb1

<400> SEQENCE: 12

atgttttcca gactgccaac atcattggcc agaaatgttg cacgtcgtgc cccaacttct 60

tttgtaagac cctctgcagc agcagcagca ttgagattct catcaacaaa gacgatgacc 120

gtcagagagg ccttgaatag tgccatggcg gaagaattgg accgtgatga tgatgtcttc 180

cttattggtg aagaagttgc acaatataac ggggcttata aggtgtcaaa gggtttattg 240

gacaggttcg gtgaacgtcg tgtggttgac acacctatta ccgaatacgg gttcacaggt 300

ttggccgttg gtgccgcttt gaagggtttg aagccaattg tagagtttat gtcgttcaat 360

ttctctatgc aagctatcga tcatgttgtc aattccgctg caaagactca ctacatgtct 420

ggtggtactc aaaaatgtca aatggtcttc agaggtccta atggtgctgc agtgggtctt 480

ggtgctcaac attcacagga cttttctcct tggtacggtt ccattccagg gttaaaggtc 540

cttgtccctt attctgctga agatgctagg ggtttgttaa aggccgccat cagagatcca 600

aaccctgttg tatttttaga gaacgaattg ttgtacggtg aatcttttga aatctcagaa 660

gaagctttat cccctgagtt caccttgcca tacaaggcta agatcgaaag agaaggtacc 720

gatatttcca ttgttacgta cacaagaaac gttcagtttt ctttggaagc cgctgaaatt 780

ctacaaaaga aatatggtgt ctctgcagaa gttatcaact tgcgttctat tagaccttta 840

gatactgaag ctatcatcaa aactgtcaag aagacaaacc acttgattac tgttgaatcc 900

actttcccat catttggtgt tggtgctgaa attgtcgccc aagttatgga gtctgaagcc 960

tttgattact tggatgctcc aatccaaaga gttactggtg ccgatgttcc aacaccttac 1020

gctaaagaat tagaagattt cgctttccct gatactccaa ccatcgttaa agctgtcaaa 1080

gaagtcttgt caattgaata a 1101

<210> SEQ ID NO: 13

<211> LENGTH: 1449

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized lat1

<400> SEQENCE: 13

atgtctgcct ttgtcagggt ggttccaaga atatccagaa gttcagtact caccagatca 60

ttgagactgc aattgagatg ctacgcatcg tacccagagc acaccattat tggtatgccg 120

gcactgtctc ctacgatgac gcaaggtaat cttgctgctt ggactaagaa ggaaggtgac 180

caattgtctc ccggtgaagt tattgccgaa atagaaacag acaaggctca aatggacttt 240

gagttccaag aagatggtta cttagccaag attctagttc ctgaaggtac aaaggacatt 300

cctgtcaaca agcctattgc cgtctatgtg gaggacaaag ctgatgtgcc agcttttaag 360

gactttaagc tggaggattc aggttctgat tcaaagacca gtacgaaggc tcagcctgcc 420

gaaccacagg cagaaaagaa acaagaagcg ccagctgaag agaccaagac ttctgcacct 480

gaagctaaga aatctgacgt tgctgctcct caaggtagga tttttgcctc tccacttgcc 540

aagactatcg ccttggaaaa gggtatttct ttgaaggatg ttcacggcac tggaccccgc 600

ggtagaatta ccaaggctga cattgagtca tatctagaaa agtcgtctaa gcagtcttct 660

caaaccagtg gtgctgccgc cgccactcct gccgccgcta cctcaagcac tactgctggc 720

tctgctccat cgccttcttc tacagcatca tatgaggatg ttccaatttc aaccatgaga 780

agcatcattg gagaacgttt attgcaatct actcaaggca ttccatcata catcgtttcc 840

tccaagatat ccatctccaa acttttgaaa ttgagacagt ccttgaacgc tacagcaaac 900

gacaagtaca aactgtccat taatgaccta ttagtaaaag ccatcactgt tgcggctaag 960

agggtgccag atgccaatgc ctactggtta cctaatgaga acgttatccg taaattcaag 1020

aatgtcgatg tctcagtcgc tgttgccaca ccaacaggat tattgacacc aattgtcaag 1080

aattgtgagg ccaagggctt gtcgcaaatc tctaacgaaa tcaaggaact agtcaagcgt 1140

gccagaataa acaaattggc accagaggaa ttccaaggtg ggaccatttg catatccaat 1200

atgggcatga ataatgctgt taacatgttt acttcgatta tcaacccacc acagtctaca 1260

atcttggcca tcgctactgt tgaaagggtc gctgtggaag acgccgctgc tgagaacgga 1320

ttctcctttg ataaccaggt taccataaca gggacctttg atcatagaac cattgatggc 1380

gccaaaggtg cagaattcat gaaggaattg aaaactgtta ttgaaaatcc tttggaaatg 1440

ctattgtga 1449

<210> SEQ ID NO: 14

<211> LENGTH: 1500

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized lpd1

<400> SEQENCE: 14

atgttaagaa tcagatcact cctaaataat aagcgtgcct tttcgtccac agtcaggaca 60

ttgaccatta acaagtcaca tgatgtagtc atcatcggtg gtggccctgc tggttacgtg 120

gctgctatca aagctgctca attgggattt aacactgcat gtgtagaaaa aagaggcaaa 180

ttaggcggta cctgtcttaa cgttggatgt atcccctcca aagcacttct aaataattct 240

catttattcc accaaatgca tacggaagcg caaaagagag gtattgacgt caacggtgat 300

atcaaaatta acgtagcaaa cttccaaaag gctaaggatg acgctgttaa gcaattaact 360

ggaggtattg agcttctgtt caagaaaaat aaggtcacct attataaagg taatggttca 420

ttcgaagacg aaacgaagat cagagtaact cccgttgatg ggttggaagg cactgtcaag 480

gaagaccaca tactagatgt taagaacatc atagtcgcca cgggctctga agttacaccc 540

ttccccggta ttgaaataga tgaggaaaaa attgtctctt caacaggtgc tctttcgtta 600

aaggaaattc ccaaaagatt aaccatcatt ggtggaggaa tcatcggatt ggaaatgggt 660

tcagtttact ctagattagg ctccaaggtt actgtagtag aatttcaacc tcaaattggt 720

gcatctatgg acggcgaggt tgccaaagcc acccaaaagt tcttgaaaaa gcaaggtttg 780

gacttcaaat taagcaccaa agttatttct gcaaagagaa acgacgacaa gaacgtcgtc 840

gaaattgttg tagaagatac taaaacgaat aagcaagaaa atttggaagc tgaagttttg 900

ctggttgctg ttggtagaag accttacatt gctggcttag gggctgaaaa gattggatta 960

gaagtagaca aaaggggacg cctagtcatt gatgaccaat ttaattccaa gttcccacac 1020

attaaagtgg taggagatgt tacatttggt ccaatgctgg ctcacaaagc cgaagaggaa 1080

ggtattgcag ctgtcgaaat gttgaaaact ggtcacggtc atgtcaacta taacaacatt 1140

ccttcggtca tgtattctca cccagaagta gcatgggttg gtaaaaccga agagcaattg 1200

aaagaagccg gcattgacta taaaattggt aagttcccct ttgcggccaa ttcaagagcc 1260

aagaccaacc aagacactga aggtttcgtg aagattttga tcgattccaa gaccgagcgt 1320

attttggggg ctcacattat cggtccaaat gccggtgaaa tgattgctga agctggctta 1380

gccttagaat atggcgcttc cgcagaagat gttgctaggg tctgccatgc tcatcctact 1440

ttgtccgaag catttaagga agctaacatg gctgcctatg ataaagctat tcattgttga 1500

<210> SEQ ID NO: 15

<211> LENGTH: 1233

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pdx1

<400> SEQENCE: 15

atgctaagtg caatttccaa agtctccact ttaaaatcat gtacaagata tttaaccaaa 60

tgcaactatc atgcatcagc taaattactt gctgtaaaga cattttcaat gcctgcaatg 120

tctcctacta tggagaaagg ggggattgtg tcttggaaat ataaagttgg cgaaccattc 180

agcgcgggcg atgtgatatt agaagtggaa acagataaat ctcaaattga tgtggaagca 240

ctggacgatg gtaaactagc taagatcctg aaagatgaag gctctaaaga tgttgatgtt 300

ggtgaaccta ttgcttatat tgctgatgtt gatgatgatt tagctactat aaagttaccc 360

caagaggcca acaccgcaaa tgcgaaatct attgaaatta agaagccatc cgcagatagt 420

actgaagcaa cacaacaaca tttaaaaaaa gccacagtta caccaataaa aaccgttgac 480

ggcagccaag ccaatcttga acagacgcta ttaccatccg tgtcattact actggctgag 540

aacaatatat ccaaacaaaa ggctttgaag gaaattgcgc catctggttc caacggtaga 600

ctattaaagg gtgatgtgct agcataccta gggaaaatac cacaagattc ggttaacaag 660

gtaacagaat ttatcaagaa gaacgaacgt ctcgatttat cgaacattaa acctatacag 720

ctcaaaccaa aaatagccga gcaagctcaa acaaaagctg ccgacaagcc aaagattact 780

cctgtagaat ttgaagagca attagtgttc catgctcccg cctctattcc gtttgacaaa 840

ctgagtgaat cattgaactc tttcatgaaa gaagcttacc agttctcaca cggaacacca 900

ctaatggaca caaattcgaa atactttgac cctattttcg aggaccttgt caccttgagc 960

ccaagagagc caagatttaa attttcctat gacttgatgc aaattcccaa agctaataac 1020

atgcaagaca cgtacggtca agaagacata tttgacctct taacaggttc agacgcgact 1080

gcctcatcag taagacccgt tgaaaagaac ttacctgaaa aaaacgaata tatactagcg 1140

ttgaatgtta gcgtcaacaa caagaagttt aatgacgcgg aggccaaggc aaaaagattc 1200

cttgattacg taagggagtt agaatcattt tga 1233

<210> SEQ ID NO: 16

<211> LENGTH: 1116

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pdhA

<400> SEQENCE: 16

atggcaaagg ctaagaaaca aaaacctatt gactttaaag agctaatggc taaagtcgac 60

gctgatttcc caactttcca aatcttggat caagatggaa aaattgtgaa tgaagattta 120

gtacctgatt tatcggatga ggaattagtt gaattaatga cacgcatggt ttggtctcgt 180

gtgttagacc aacgttctac tgcattaaac cgtcaaggac gcttaggatt cttcgcgcca 240

acagctggac aagaagcaag ccaattggca agtcaatttg caatggaaaa agaagactac 300

ttactaccag gttaccgtga tgtacctcaa ttagtacaac atggtttacc attaagagaa 360

gctttcttat ggtctcgtgg tcacgtagca gggaactact acgcggaaga tttaaatgca 420

ttaccaccac aaattatcat tggtgctcaa tacatccaag cagctggtgt tgctttagga 480

ttgaaaaaac gtggaaaaga aaatgttgtc ttcacttata ctggtgacgg cggttcttca 540

caaggggact tctatgaagc aattaacttt gctggtgctt accaagcaaa cggtgtcttc 600

attatccaaa acaatggttt tgcgatttct acacctcgtg aaaaacaaac agcggctaaa 660

actttagctc aaaaagctgt tgcagcagga attcctggta ttcaagttga tggtatggat 720

ccattagcag tttacgcaat tgcaaaagaa gcacgtgatt ggtcagctgc aggaaacggt 780

ccagttttaa ttgaaacatt aacctatcgt tatggtccac atactttatc tggagacgat 840

ccaacacgtt accgttcaaa agaaatggat gacgaatggg tacaaaaaga tccattgact 900

cgtttccgta aatatctaac agataaaggc ttatggtctg aagcaaaaga agaagaaatt 960

attgaaaaaa caaaagaaga aatcaaagta gcgattgcag aagcggataa agcgccaaaa 1020

caaaaagttt ctgatttctt gaaaaatatg tttgaagttc aacctcaaac aattaaagaa 1080

caaattgcat tttatgaagc gaaggagtcg aaataa 1116

<210> SEQ ID NO: 17

<211> LENGTH: 735

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized adc

<400> SEQENCE: 17

atgttaaagg atgaagtaat taaacaaatt agcacgccat taacttcgcc tgcatttcct 60

agaggaccct ataaatttca taatcgtgag tattttaaca ttgtatatcg tacagatatg 120

gatgcacttc gtaaagttgt gccagagcct ttagaaattg atgagccctt agtcaggttt 180

gaaattatgg caatgcatga tacgagtgga cttggttgtt atacagaaag cggacaggct 240

attcccgtaa gctttaatgg agttaaggga gattatcttc atatgatgta tttagataat 300

gagcctgcaa ttgcagtagg aagggaatta agtgcatatc ctaaaaagct cgggtatcca 360

aagctttttg tggattcaga tactttagta ggaactttag actatggaaa acttagagtt 420

gcgacagcta caatggggta caaacataaa gccttagatg ctaatgaagc aaaggatcaa 480

atttgtcgcc ctaattatat gttgaaaata atacccaatt atgatggaag ccctagaata 540

tgtgagctta taaatgcgaa aatcacagat gttaccgtac atgaagcttg gacaggacca 600

actcgactgc agttatttga tcacgctatg gcgccactta atgatttgcc agtaaaagag 660

attgtttcta gctctcacat tcttgcagat ataatattgc ctagagctga agttatatat 720

gattatctta agtaa 735

<210> SEQ ID NO: 18

<211> LENGTH: 741

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized adc

<400> SEQENCE: 18

atgttagaaa gtgaagtatc taaacaaatt acaactccac ttgctgctcc agcgtttcct 60

agaggaccat ataggtttca caatagagaa tatctaaaca ttatttatcg aactgattta 120

gatgctcttc gaaaaatagt accagagcca cttgaattag atagagcata tgttagattt 180

gaaatgatgg ctatgcctga tacaaccgga ctaggctcat atacagaatg tggtcaagct 240

attccagtaa aatataatgg tgttaagggt gactacttgc atatgatgta tctagataat 300

gaacctgcta ttgctgttgg aagagaaagt agcgcttatc caaaaaagct tggctatcca 360

aagctatttg ttgattcaga tactttagtt gggacactta aatatggtac attaccagta 420

gctactgcaa caatgggata taagcacgag cctctagatc ttaaagaagc ctatgctcaa 480

attgcaagac ccaattttat gctaaaaatc attcaaggtt acgatggtaa gccaagaatt 540

tgtgaactaa tatgtgcaga aaatactgat ataactattc acggtgcttg gactggaagt 600

gcacgtctac aattatttag ccatgcacta gctcctcttg ctgatttacc tgtattagag 660

attgtatcag catctcatat cctcacagat ttaactcttg gaacacctaa ggttgtacat 720

gattatcttt cagtaaaata a 741

<210> SEQ ID NO: 19

<211> LENGTH: 1056

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized adh

<400> SEQENCE: 19

atgaaaggtt ttgcaatgct aggtattaat aagttaggat ggatcgaaaa agaaaggcca 60

gttgcgggtt catatgatgc tattgtacgc ccattagcag tatctccgtg tacatcagat 120

atacatactg tttttgaggg agctcttgga gataggaaga atatgatttt agggcatgaa 180

gctgtaggtg aagttgttga agtaggaagt gaagtgaagg attttaaacc tggtgacaga 240

gttatagttc cttgtacaac tccagattgg agatctttgg aagttcaagc tggttttcaa 300

cagcactcaa acggtatgct cgcaggatgg aaattttcaa atttcaagga tggagttttt 360

ggtgaatatt ttcatgtaaa tgatgcggat atgaatcttg cgattctacc taaagacatg 420

ccattagaaa atgctgttat gataacagat atgatgacta ctggatttca tggagcagaa 480

cttgcagata ttcaaatggg ttcaagtgtt gtggtaattg gcattggagc tgttggctta 540

atgggaatag caggtgctaa attacgtgga gcaggtagaa taattggagt ggggagcagg 600

ccgatttgtg ttgaggctgc aaaattttat ggagcaacag atattctaaa ttataaaaat 660

ggtcatatag ttgatcaagt tatgaaatta acgaatggaa aaggcgttga ccgcgtaatt 720

atggcaggcg gtggttctga aacattatcc caagcagtat ctatggttaa accaggagga 780

ataatttcta atataaatta tcatggaagt ggagatgctt tactaatacc acgtgtagaa 840

tggggatgtg gaatggctca caagactata aaaggaggtc tttgtcctgg gggacgtttg 900

agagcagaaa tgttaagaga tatggtagta tataatcgtg ttgatctaag taaattagtt 960

acacatgtat atcatggatt tgatcacata gaagaagcac tgttattaat gaaagacaag 1020

ccaaaagact taattaaagc agtagttata ttataa 1056

<210> SEQ ID NO: 20

<211> LENGTH: 414

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized mgsA

<400> SEQENCE: 20

atgaaaattg ctttgatcgc gcatgacaag aaaaaacagg atatggttca atttacgact 60

gcctatcggg atattttaaa gaatcatgat ctatacgcaa ccggaaccac agggttgaaa 120

attcatgagg cgacaggtct tcaaattgaa cgttttcaat ccggcccttt agggggagac 180

cagcaaatcg gtgcactgat cgctgccaat gcactcgatc ttgtcatttt tttgcgcgac 240

ccgctgaccg cgcagccgca tgaaccggat gtctcggcat taatccgttt atgtgatgtg 300

tattccattc cgctcgccac aaatatgggt actgcggaaa ttcttgtgcg cacacttgat 360

gaaggtgttt tcgaattccg tgaccttctt cggggagaag agccgaatgt ataa 414

<210> SEQ ID NO: 21

<211> LENGTH: 459

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized mgsA

<400> SEQENCE: 21

atggaactga cgactcgcac tttacctgcg cggaaacata ttgcgctggt ggcacacgat 60

cactgcaaac aaatgctgat gagctgggtg gaacggcatc aaccgttact ggaacaacac 120

gtactgtatg caacaggcac taccggtaac ttaatttccc gcgcgaccgg catgaacgtc 180

aacgcgatgt tgagtggccc aatggggggt gaccagcagg ttggcgcatt gatctcagaa 240

gggaaaattg atgtattgat tttcttctgg gatccactaa atgccgtgcc gcacgatcct 300

gacgtgaaag ccttgctgcg tctggcgacg gtatggaaca ttccggtcgc caccaacgtg 360

gcaacggcag acttcataat ccagtcgccg catttcaacg acgcggtcga tattctgatc 420

cccgattatc agcgttatct cgcggaccgt ctgaagtaa 459

<210> SEQ ID NO: 22

<211> LENGTH: 459

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized mgsA

<400> SEQENCE: 22

atggaactga cgactcgcac tttacctgcg cggaaacata ttgcgctggt ggcacacgat 60

caatgcaaac aaatgctgat gagctgggtg gaacggcatc aaccgttact ggaacaacac 120

gtactgtatg caacaggcac taccggtaac ttaatttccc gcgcgaccgg catgaacgtc 180

aacgcgatgt tgagtggccc aatggggggt gaccagcagg ttggcgcatt gatctcagaa 240

gggaaaattg atgtattgat tttcttctgg gatccactaa atgccgtgcc gcacgatcct 300

gacgtgaaag ccttgctgcg tctggcgacg gtatggaaca ttccggtcgc caccaacgtg 360

gcaacggcag acttcataat ccagtcgccg catttcaacg acgcggtcga tattctgatc 420

cccgattatc agcgttatct cgcggaccgt ctgaagtaa 459

<210> SEQ ID NO: 23

<211> LENGTH: 981

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ydjg

<400> SEQENCE: 23

atgaaaaaga tacctttagg cacaacggat attacgcttt cgcgaatggg gttggggaca 60

tgggccattg gcggcggtcc tgcatggaat ggcgatctcg atcggcaaat atgtattgat 120

acgattcttg aagcccatcg ttgtggcatt aatctgattg atactgcgcc aggatataac 180

tttggcaata gtgaagttat cgtcggtcag gcgttaaaaa aactgccccg tgaacaggtt 240

gtagtagaaa ccaaatgcgg cattgtctgg gaacgaaaag gaagtttatt caacaaagtt 300

ggcgatcggc agttgtataa aaacctttcc ccggaatcta tccgcgaaga ggtagcagcg 360

agcttgcaac gtctgggtat tgattacatc gatatctaca tgacgcactg gcagtcggtg 420

ccgccatttt ttacgccgat cgctgaaact gtcgcagtgc ttaatgagtt aaagtctgaa 480

gggaaaattc gcgctatagg cgctgctaac gtcgatgctg accatatccg cgagtatctg 540

caatatggtg aactggatat tattcaggcg aaatacagta tcctcgaccg ggcaatggaa 600

aacgaactgc tgccactatg tcgtgataat ggcattgtgg ttcaggttta ttccccgcta 660

gagcagggat tgttgaccgg caccatcact cgtgattacg ttccgggcgg cgctcgggca 720

aataaagtct ggttccagcg tgaaaacatg ctgaaagtga ttgatatgct tgaacagtgg 780

cagccacttt gtgctcgtta tcagtgcaca attcccactc tggcactggc gtggatatta 840

aaacagagtg atttaatctc cattcttagt ggggctactg caccggaaca ggtacgcgaa 900

aatgtcgcgg cactgaatat caacttatcg gatgcagacg caacattgat gagggaaatg 960

gcagaggccc tggagcgtta a 981

<210> SEQ ID NO: 24

<211> LENGTH: 939

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ypr1

<400> SEQENCE: 24

atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60

ccagtgttgg gtttcggcac ttggcgttcc gttgacaata gcggttacca ttctgtaatt 120

gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180

gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240

aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300

ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360

agagttactg atggtaacgt tctgtgtatt ccaacattag aagatggcac tgttgacatc 420

gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagaatt gccaaagacg 480

ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540

gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600

ccacaagacg aattgattgc cttttgtaaa gaaaagggta tcgttgttga agcctactca 660

ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720

aagcacggtg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780

gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840

cccgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900

gttgatatga agtggggatc cttcccaatt ttccaatga 939

<210> SEQ ID NO: 25

<211> LENGTH: 771

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized budC

<400> SEQENCE: 25

atgaaaaaag tcgcacttgt taccggcgcc ggccagggga ttggtaaagc tatcgccctt 60

cgtctggtga aggatggatt tgccgtggcc attgccgatt ataacgacgc caccgccaaa 120

gcggtcgcct ccgaaatcaa ccaggccggc ggccgcgcca tggcggtgaa agtggatgtt 180

tctgaccgcg accaggtatt tgccgccgtc gaacaggcgc gcaaaacgct gggcggcttc 240

gacgtcatcg tcaacaacgc cggcgtggcg ccgtccacgc cgatcgagtc cattaccccg 300

gagattgtcg acaaagtcta caacatcaac gtcaaagggg tgatctgggg catccaggcg 360

gcggtcgagg cctttaagaa agagggtcac ggcgggaaaa tcatcaacgc ctgttcccag 420

gccggccacg tcggtaaccc ggagctggcg gtgtatagct cgagtaaatt cgccgtacgc 480

ggcttaaccc agaccgccgc tcgcgacctc gcgccgctgg gcatcacggt caacggctac 540

tgcccgggga ttgtcaaaac gccaatgtgg gccgaaattg accgccaggt gtccgaagcc 600

gccggtaaac cgctgggcta cggtaccgcc gagttcgcca aacgcatcac tctcggtcgt 660

ctgtccgagc cggaagatgt cgccgcctgc gtctcctatc ttgccagccc ggattctgat 720

tacatgaccg gtcagtcgtt gctgatcgac ggcgggatgg tatttaacta a 771

<210> SEQ ID NO: 26

<211> LENGTH: 1149

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized fucO

<400> SEQENCE: 26

atggctaaca gaatgattct gaacgaaacg gcatggtttg gtcggggtgc tgttggggct 60

ttaaccgatg aggtgaaacg ccgtggttat cagaaggcgc tgatcgtcac cgataaaacg 120

ctggtgcaat gcggcgtggt ggcgaaagtg accgataaga tggatgctgc agggctggca 180

tgggcgattt acgacggcgt agtgcccaac ccaacaatta ctgtcgtcaa agaagggctc 240

ggtgtattcc agaatagcgg cgcggattac ctgatcgcta ttggtggtgg ttctccacag 300

gatacttgta aagcgattgg cattatcagc aacaacccgg agtttgccga tgtgcgtagc 360

ctggaagggc tttccccgac caataaaccc agtgtaccga ttctggcaat tcctaccaca 420

gcaggtactg cggcagaagt gaccattaac tacgtgatca ctgacgaaga gaaacggcgc 480

aagtttgttt gcgttgatcc gcatgatatc ccgcaggtgg cgtttattga cgctgacatg 540

atggatggta tgcctccagc gctgaaagct gcgacgggtg tcgatgcgct cactcatgct 600

attgaggggt atattacccg tggcgcgtgg gcgctaaccg atgcactgca cattaaagcg 660

attgaaatca ttgctggggc gctgcgagga tcggttgctg gtgataagga tgccggagaa 720

gaaatggcgc tcgggcagta tgttgcgggt atgggcttct cgaatgttgg gttagggttg 780

gtgcatggta tggcgcatcc actgggcgcg ttttataaca ctccacacgg tgttgcgaac 840

gccatcctgt taccgcatgt catgcgttat aacgctgact ttaccggtga gaagtaccgc 900

gatatcgcgc gcgttatggg cgtgaaagtg gaaggtatga gcctggaaga ggcgcgtaat 960

gccgctgttg aagcggtgtt tgctctcaac cgtgatgtcg gtattccgcc acatttgcgt 1020

gatgttggtg tacgcaagga agacattccg gcactggcgc aggcggcact ggatgatgtt 1080

tgtaccggtg gcaacccgcg tgaagcaacg cttgaggata ttgtagagct ttaccatacc 1140

gcctggtaa 1149

<210> SEQ ID NO: 27

<211> LENGTH: 804

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized yafB

<400> SEQENCE: 27

atggctatcc ctgcatttgg tttaggtact ttccgtctga aagacgacgt tgttatttca 60

tctgtgataa cggcgcttga acttggttat cgcgcaattg ataccgcaca aatctatgat 120

aacgaagccg cagtaggtca ggcgattgca gaaagtggcg tgccacgtca tgaactctac 180

atcaccacta aaatctggat tgaaaatctc agcaaagaca aattgatccc aagtctgaaa 240

gagagcctgc aaaaattgcg taccgattat gttgatctga cgctaatcca ctggccgtca 300

ccaaacgatg aagtctctgt tgaagagttt atgcaggcgc tgctggaagc caaaaaacaa 360

gggctgacgc gtgagatcgg tatttccaac ttcacgatcc cgttgatgga aaaagcgatt 420

gctgctgttg gtgctgaaaa catcgctact aaccagattg aactctctcc ttatctgcaa 480

aaccgtaaag tggttgcctg ggctaaacag cacggcatcc atattacttc ctatatgacg 540

ctggcgtatg gtaaggccct gaaagatgag gttattgctc gtatcgcagc taaacacaat 600

gcgactccgg cacaagtgat tctggcgtgg gctatggggg aaggttactc agtaattcct 660

tcttctacta aacgtaaaaa cctggaaagt aatcttaagg cacaaaattt acagcttgat 720

gccgaagata aaaaagcgat cgccgcactg gattgcaacg accgcctggt tagcccggaa 780

ggtctggctc ctgaatggga ttaa 804

<210> SEQ ID NO: 28

<211> LENGTH: 2364

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized dhaB1

<400> SEQENCE: 28

atgataagta aaggatttag tacccaaaca gaaagaataa atattttaaa ggctcaaata 60

ttaaatgcta aaccatgtgt tgaatcagaa agagcaatat taataacaga atcatttaaa 120

caaacagaag gccagccagc aattttaaga agagcattgg cattgaaaca catacttgaa 180

aatatcccta taacaattag agatcaagaa cttatagtgg gaagtttaac taaagaacca 240

aggtcttcac aagtatttcc tgagttttct aataagtggt tacaagatga attggataga 300

ttaaataaga gaactggaga tgcattccaa atttcagaag aaagtaaaga aaaattaaaa 360

gatgtctttg agtattggaa tggaaagaca acaagtgagt tagcaacttc atatatgaca 420

gaggaaacaa gagaggcagt aaattgtgat gtatttactg taggaaacta ctattataat 480

ggcgtaggac atgtatctgt agattatgga aaagtattaa gggttggatt taatgggatt 540

ataaatgagg ctaaggaaca attagaaaaa aacaggagta tagatcctga ttttataaag 600

aaagaaaaat tcctaaatag tgttattatc tcatgcgaag ctgcaataac atatgtaaat 660

agatatgcta aaaaggctaa agagattgca gataatacaa gtgatgcaaa aagaaaagct 720

gaattaaatg aaatagcaaa aatttgttca aaagtttcag gagagggagc taaatctttc 780

tatgaagcat gtcaattatt ttggtttatt catgcaataa taaatataga atctaatgga 840

cattctattt ctccagctag atttgatcaa tacatgtatc catattatga aaatgataaa 900

aatataacag ataagtttgc tcaagaatta atagattgta tctggattaa attaaatgat 960

attaataaag taagagatga gatttcaact aaacattttg gtggttaccc aatgtatcaa 1020

aacttaattg ttgggggtca aaattcagaa ggaaaagatg caactaataa agtatcatat 1080

atggcattag aagcagctgt ccatgtaaag ttgcctcagc catctttgtc agtaagaata 1140

tggaataaga ctccagatga atttttgctt agagcagcag aattaactag agaagggtta 1200

ggacttcctg cttattataa tgatgaagtt attattccag cattagtttc tagaggtctt 1260

acattagaag atgcaagaga ctacggaata attggatgtg ttgaaccaca aaagccagga 1320

aaaacagaag gatggcatga ttcagcattc tttaatcttg caagaatagt agagttaact 1380

ataaattctg gatttgataa aaataaacag attggaccta aaactcaaaa ttttgaagaa 1440

atgaaatcct ttgatgaatt catgaaagct tataaagctc aaatggagta ttttgtaaaa 1500

catatgtgct gtgctgataa ttgcatagat attgcacatg cagaaagagc tccattacct 1560

ttcttgtcat caatggttga taattgtatc ggaaaaggaa agagccttca agatggtggt 1620

gcagaatata acttcagtgg accacaaggt gttggagtag ctaatattgg agattcatta 1680

gttgcagtta aaaaaattgt gtttgatgaa aataagatta ctccttcaga attaaagaaa 1740

acattaaata atgattttaa aaattcagaa gaaatacaag ccttactaaa aaatgctcct 1800

aagtttggaa atgatattga tgaagttgat aatttagcta gagagggtgc attagtatac 1860

tgtagagaag ttaataaata tacaaatcca aggggaggaa attttcaacc aggattatat 1920

ccatcttcaa ttaatgtata ttttggaagc ttaacaggtg ctactccaga tggaaggaaa 1980

tccggacaac cattagctga tggggtttct ccatcaagag gctgtgatgt atctggacct 2040

actgcagctt gtaactcagt tagtaaatta gatcatttta tagcttcaaa tggaacttta 2100

tttaatcaaa aattccatcc gtcagcatta aaaggtgata atggattaat gaatttatca 2160

tcattaataa gaagttattt tgatcaaaag ggatttcatg ttcaatttaa tgtaatagat 2220

aaaaaaatat tacttgcagc acaaaaaaat cctgaaaaat atcaagattt aattgttaga 2280

gttgcaggat atagtgcaca gttcatttct ttagataaat ctattcaaaa tgatattatt 2340

gcaagaactg aacatgttat gtaa 2364

<210> SEQ ID NO: 29

<211> LENGTH: 915

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized dhaB2

<400> SEQENCE: 29

atgagtaagg agataaaagg cgttttattt aacatacaaa aattttcgtt acatgatggg 60

cctggaataa gaactatagt attttttaag ggatgttcaa tgtcgtgctt atggtgcagt 120

aatccagaat cccaagatat taaacctcaa gtaatgttta ataaaaattt atgtacaaaa 180

tgtggaagat gtaaatctca atgtaaaagt gcagctattg atatgaattc agaatatagg 240

atagataaaa gcaaatgtac agagtgtaca aaatgtgttg ataattgctt aagcggggca 300

cttgttattg aaggaaggaa ttacagtgtt gaagacgtta taaaggaatt gaaaaaagat 360

agtgttcaat atagaagatc aaacggtgga attacactat ctggagggga agtattactt 420

caaccagatt ttgcagtgga gcttttaaaa gagtgtaaat catatggctg gcacactgcc 480

attgaaacag caatgtatgt taatagtgaa tctgtaaaaa aagtaattcc atatatagat 540

ctggctatga ttgatataaa aagtatgaat gatgaaatcc ataggaaatt tacaggagtg 600

agtaacgaaa taatattaca aaacattaaa ttaagtgatg aattagctaa agaaataata 660

atcagaattc ctgtaataga aggatttaat gcagatttac aaagtatagg agcaatagct 720

caattttcaa aatcattaac aaatcttaaa agaatagatc ttcttccata ccataattat 780

ggagaaaata agtatcaagc aattggaaga gagtattctt tgaaagaact aaaatcacct 840

agtaaagaca aaatggaaag attaaaagct ttagttgaaa tcatgggaat accgtgcaca 900

attggagctg agtaa 915

<210> SEQ ID NO: 30

<211> LENGTH: 2532

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized b1

<400> SEQENCE: 30

atgggaaatt atgatagtac tccaattgcg aagtcggatc gtataaaaag acttgtagat 60

catctgtatg caaagatgcc tgagattgag gcggcaagag cggaactgat cacagaatca 120

tttaaggcta cggaaggtca gccggtagtg atgcgcaaag cacgtgcttt tgaacatatt 180

ttaaagaatc ttccgatcat tatcagacca gaagaattaa ttgtcggaag tacaacgatc 240

gcaccgagag gatgccagac atatccggaa ttttcatatg aatggttaga ggcagaattc 300

gaaacagtcg aaacaagaag tgctgatcca ttctatattt cagaggaaac aaaaaagaga 360

ttattagctg cagatgctta ctggaaagga aaaacaacca gtgagctggc aacttcctat 420

atggctccgg agacactccg tgccatgaaa cataatttct ttacaccggg caactatttt 480

tataatggtg taggacatgt aacagttcag tatgaaaccg tattggcgat cggtctgaat 540

ggtgtaaaag aaaaagtcag aaaagagatg gagaactgcc attttggaga tgcggattat 600

tctaccaaga tgtgtttctt agaatccatc ctgatttcct gtgatgcagt catcacttat 660

gcaaatcgtt atgcgaaaat ggcagaagag atggcagaga aagaaacaga tgcagcaaga 720

agacaggagc ttctgacaat tgcaagagta tgtaaaaatg taccggaatt ccctgctgaa 780

agcttccagg aggcgtgcca gtccttctgg ttcatccagc aggtattaca gattgaatcc 840

agtggacatt ctatttcacc gggacgtttt gaccagtata tgtatcctta ttacgagaag 900

gatttaaaag aaggcagtct cacccgtgag tacgcacagg aactgatcga ctgtatctgg 960

gtaaaattaa atgatctgaa taaatgtcgt gatgccgcaa gtgcagaagg ttttgcagga 1020

tattccttat tccagaacct gatcgttggt ggacagacag ttcagggaag agacgctacc 1080

aatgatcttt cgtttatgtg catcactgcc agtgagcatg tatttttacc aatgccatcc 1140

ttatcgatcc gtgtgtggca tggatcatcc aaggcattat taatgcgtgc ggcagagctg 1200

acaagaaccg gtatcggttt accggcttat tataatgacg aagttatcat tcctgcattg 1260

gttcatcgtg gagcaaccat ggacgaggca aggaattaca acatcatcgg atgtgtagaa 1320

ccgcaggttc cgggtaaaac agacggatgg cacgatgcag cgttcttcaa tatgtgccgc 1380

ccattggaga tggtattttc caatggttat gacaatggag agatcgcaag tatccagacc 1440

ggtaatgtgg agagcttcca gtcatttgat gaatttatgg aagcatacag aaaacagatg 1500

ttatataaca tcgaattgat ggtaaatgca gataatgcaa ttgattatgc tcatgcaaag 1560

cttgcaccat taccatttga gtcatgtctg gtagatgact gcatcaagcg gggaatgagt 1620

gcacaggaag gcggagcaat ttataacttt accggtccgc agggctttgg tatcgcaaat 1680

gtcgcagact ctttatatac gatcaagaag ctggtatttg aagaaaaacg cattaccatg 1740

ggcgagttaa agaaagctct tgagatgaat tacggtaaag ggctggatgc cacaactgcc 1800

ggagatattg caatgcaggt tgcaaaagga ttaaaagatg caggtcagga agtgggacct 1860

gatgtgatag cgaatacgat cagacaggta ttagagatgg aattaccgga agatgtcagg 1920

aagcgttatg aagagatcca tgaaatgatc cttgaacttc cgaaatacgg aaatgatatt 1980

gatgaagtag atgagcttgc ccgcgaggca gcatatttct acacaagacc attagagaca 2040

ttcaaaaatc caagaggtgg aatgtatcag gcaggtctct atccggtatc agccaatgtt 2100

ccattaggag ctcagaccgg tgctactccg gacggaagat tagcacatac tccggtggca 2160

gatggagtcg gaccgacatc aggattcgat atcagtggac cgacagcatc ctgtaactca 2220

gttgcaaaat tagatcatgc gatcgcaagt aacggaacac tctttaatat gaaaatgcat 2280

ccaacagcta tggctggaga gaaggggctg gagagcttta tttctctgat tcgtggttac 2340

tttgatcagc agggtatgca catgcagttt aatgtcgtag accgtgcaac tcttttggac 2400

gcacaggctc atccagaaaa atacagtggg ctgatcgtac gtgtagccgg atattctgct 2460

ttgtttacta cgttatcgaa atccttacag gatgatatca ttaagagaac agaacaggct 2520

gataatcgat ag 2532

<210> SEQ ID NO: 31

<211> LENGTH: 795

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized b2

<400> SEQENCE: 31

atgaaagaat atttgaatac atccggcagg atttttgata ttcaaagata ttccatacat 60

gatggtccgg gagtccgaac catagtcttc ttaaaaggat gtgcgttacg atgcagatgg 120

tgctgtaatc cggaatcaca gtcttttgaa gtggaaacaa tgacgatcaa cggaaaacca 180

aaggttatgg gcaaagatgt aactgtagcg gaggttatga agacagtaga aagagacatg 240

ccttattatt tacagtccgg tggaggaatc actctttccg gtggtgaatg tacgcttcaa 300

ccggagttct cattagggct tttaagagca gcaaaagatt tgggaatatc aacagccatt 360

gaaagtatgg cttatgcaaa atacgaagtg atcgaaacac tgcttccgta tctggatact 420

tacttaatgg atattaagca tatgaatccg gaaaagcata aagaatatac aggtcatgat 480

aatctcagaa tgttggagaa tgcactcagg gtagcccaca gtgggcagac agaactgatc 540

atccgtgttc ctgttattcc tggatttaat gctacagagc aggaattgct tgatatagcg 600

aagtttgcag ataccttacc gggcgtcaga cagatccaca tattgcctta tcataacttt 660

ggtcagggaa aatacgaagg attgaacaga gactatccaa tgggagatac agagaagcct 720

tccaatgagc agatgaaggc atttcaggaa atgatccaaa aaaatacgtc attacactgc 780

cagattggtg gttaa 795

<210> SEQ ID NO: 32

<211> LENGTH: 2580

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized adh

<400> SEQENCE: 32

atgaaggtaa ctaatgttga agaactgatg aaaaaaatgc aggaagtgca aaatgctcaa 60

aaaaaatttg ggagttttac tcaggaacaa gtagatgaaa ttttcaggca agcagcacta 120

gcagctaaca gtgccagaat agatctagct aaaatggcag tggaagaaac taaaatggga 180

attgtagagg ataaggttat aaaaaatcat tttgttgcag aatacatata taataagtat 240

aaaaatgaaa aaacttgtgg gattttggaa gaagatgaag gctttggaat ggttaaaatt 300

gcagaacctg taggtgtgat tgcagcagta attccaacaa caaatccaac atctacagca 360

atatttaaag cattattagc tttgaaaaca agaaatggta taattttttc accacatcca 420

agagcaaaaa agtgtactat tgcagcagct aagttagttc ttgatgctgc agttaaagca 480

ggtgctccta aaggaattat aggttggata gatgaacctt ctattgaact ttcacagata 540

gtaatgaaag aagctgatat aatccttgca acaggtggtc caggtatggt taaagcagct 600

tattcttcag gtaaacctgc tataggggtt ggtcctggta acacacctgc tttaattgat 660

gaaagtgctg atattaaaat ggcagtaaat tcaatacttc tttccaaaac ttttgataat 720

ggtatgattt gtgcttcaga gcagtcggta gtagttgtag attcaatata tgaagaagtt 780

aagaaagaat ttgctcatag aggagcttat attttaagta aggatgaaac aactaaagtt 840

ggaaaaatac tcttagttaa tggtacatta aatgctggta tcgttggtca gagtgcttat 900

aaaatagcag aaatggcagg agttaaagtt ccagaagatg ctaaagttct tataggagaa 960

gtaaaatcag tggagcattc agaagagcca ttttcacatg aaaagttatc tccagtttta 1020

gctatgtata gagctaaaaa ttttgatgaa gctcttttaa aagctggaag attagttgaa 1080

ctcggtggaa tgggtcatac atctgtatta tatgtaaatg caataactga aaaagtaaaa 1140

gtagaaaaat ttagagaaac tatgaagact ggtagaacat taataaatat gccttcagca 1200

caaggtgcta taggagacat atataacttt aaactagctc cttcattaac attaggttgt 1260

ggttcatggg gaggaaactc cgtatcagaa aatgttggac ctaaacactt attaaatata 1320

aaaagtgttg ctgagaggag agaaaatatg ctttggttta gagttcctga aaaggtttat 1380

tttaaatatg gtagtcttgg agttgcatta aaagaattag atattttgga taagaaaaaa 1440

gtatttatag taacagataa agttctttat caattaggtt atatagatag agttacaaag 1500

attcttgaag aattgaaaat ttcatataaa atatttacag atgtagaacc agatccaacc 1560

ctagctacag ctaaaaaagg tgcagaagaa ttgttatcat ttaatccaga tactattata 1620

gcagttggtg gtggttcagc aatggatgct gctaagatta tgtgggtaat gtatgaacat 1680

ccggaagtaa gatttgaaga tttagctatg agatttatgg atataagaaa gagagtatat 1740

acttttccta agatgggtga aaaagcaatg atgatttctg ttgcaacatc agcaggaaca 1800

ggatcagaag taacaccttt tgcagtaatt actgatgaaa aaacaggagc taaatatcca 1860

ttagctgatt atgaattaac tccaaatatg gctataattg atgctgaact tatgatgggt 1920

atgccaaaag gattaacagc agcttcagga atagatgcac taactcatgc aatagaagct 1980

tatgtatcaa taatggcttc agaatatact aatggattag cgttagaagc aataagattg 2040

atatttaagt atttaccaat agcttacagt gaaggaacaa caagtataaa ggcaagagaa 2100

aaaatggcgc atgcttcaac aatagctggt atggcatttg ctaatgcatt tttaggagta 2160

tgtcattcaa tggcacataa attaggatca actcatcacg taccacatgg cattgccaat 2220

gcactactta taaatgaagt tataaaattt aatgcagtag aaaatccaag aaaacaagct 2280

gcatttccac aatataagta tccaaatata aaaaagagat atgctagaat agcagattac 2340

cttaacttag gtgggtcaac agacgatgaa aaagtacaat tattaataaa tgctatagat 2400

gaattaaaag ctaagataaa tattccagaa agtattaaag aagcaggagt aacagaagaa 2460

aaattttatg ctactttaga taaaatgtca gaattagctt ttgatgatca atgtacaggt 2520

gcaaacccta gatatccatt aataagtgaa ataaaacaaa tgtatgtaaa tgcattttaa 2580

<210> SEQ ID NO: 33

<211> LENGTH: 990

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ldhA

<400> SEQENCE: 33

atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60

gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120

actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180

ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240

aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300

gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360

caccgcgcgt atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt 420

actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480

cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540

gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600

atctctctgc actgcccgct gacaccggaa aactatcatc tgttgaacga agccgccttc 660

gaacagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720

caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780

gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtgatcca ggatgacgta 840

ttccgtcgcc tgtctgcctg ccacaacgtg ctgtttaccg ggcaccaggc attcctgaca 900

gcagaagcac tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960

ggcgaaacct gcccgaacga actggtttaa 990

<210> SEQ ID NO: 34

<211> LENGTH: 930

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ldhL2

<400> SEQENCE: 34

atggataaga agcaacgcaa agtcgtaatt gttggtgatg gctcggtggg ttcatcattt 60

gccttttcat tggtccaaaa ttgcgcccta gatgaactcg ttatcgttga cttggttaaa 120

acgcacgcag agggggacgt taaggatttg gaagatgttg ccgcctttac gaatgcgacc 180

aacattcata ccggtgaata tgcggatgcg cgtgatgctg acatcgttgt cattacggct 240

ggtgtgcctc gtaagcctgg tgagagtcgt ttagatttga ttaaccgcaa tacgaagatt 300

ctggaatcca tcgtcaaacc agtggttgcg agtggtttta atggttgctt cgttatctca 360

agtaatcccg tcgatatttt gacttcgatg acgcaacgtt tatccggttt tccacggcat 420

cgggtcattg gtaccgggac ttccttggat acggcgcggt tacgggtcgc cttggctcag 480

aagttgaatg ttgccaccac tgcagttgat gctgcggtac ttggagaaca tggtgatagt 540

tccatcgtta attttgatga aattatgatc aatgctcagc ccttaaagac ggtcacaacg 600

gtcgatgatc agttcaaagc tgaaatcgag caagctgttc gtggtaaagg tggtcaaatc 660

attagtcaga agggggccac gttctatggg gtcgccgtta gtttgatgca aatctgccga 720

gcaattttga acgatgaaaa tgctgagttg attgtctccg ccgctttgtc tggtcaatat 780

ggcattaacg atttgtactt ggggtcaccc gccattatta accgcaacgg gctccaaaaa 840

gtgatcgaag ctgagctatc agatgatgag cgtgcccgga tgcaacattt cgcagccaag 900

atgctgacca tgatgaatgt ggcatcataa 930

<210> SEQ ID NO: 35

<211> LENGTH: 999

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ldh2

<400> SEQENCE: 35

atggcaactc tcaaggatca gctgattcag aatcttctta aggaagaaca tgtcccccag 60

aataagatta caattgttgg ggttggtgct gttggcatgg cctgtgccat cagtatctta 120

atgaaggact tggcagatga agttgctctt gttgatgtca tggaagataa actgaaggga 180

gagatgatgg atctccaaca tggcagcctt ttccttagaa caccaaaaat tgtctctggc 240

aaagactata atgtgacagc aaactccagg ctggttatta tcacagctgg ggcacgtcag 300

caagagggag agagccgtct gaatttggtc cagcgtaacg tgaacatctt taaattcatc 360

attcctaata ttgtaaaata cagcccaaat tgcaagttgc ttgttgtttc caatccagtc 420

gatattttga cctatgtggc ttggaagata agtggctttc ccaaaaaccg tgttattgga 480

agtggttgca atctggattc agctcgcttc cgttatctca tgggggagag gctgggagtt 540

cacccattaa gctgccatgg gtggatcctt ggggagcatg gtgactctag tgtgcctgta 600

tggagtggag tgaatgttgc tggtgtctcc ctgaagaatt tacaccctga attaggcact 660

gatgcagata aggaacagtg gaaagcggtt cacaaacaag tggttgacag tgcttatgag 720

gtgatcaaac tgaaaggcta cacatcctgg gccattggac tgtcagtggc cgatttggca 780

gaaagtataa tgaagaatct taggcgggtg catccgattt ccaccatgat taagggtctc 840

tatggaataa aagaggatgt cttccttagt gttccttgca tcttgggaca gaatggaatc 900

tcagacgttg tgaaagtgac tctgactcat gaagaagagg cctgtttgaa gaagagtgca 960

gatacacttt gggggatcca gaaagaactg cagttttaa 999

<210> SEQ ID NO: 36

<211> LENGTH: 1575

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pct

<400> SEQENCE: 36

atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60

acagttacaa caagtggttt cgttggaaat gcaatccctg aggctcttga tagagctgta 120

gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta catatgttta ttgtggttct 180

caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240

tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300

atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360

cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420

ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480

caggaatatt tattctaccc tgcttttcct attcatgtag ctcttattcg tggtacttac 540

gctgatgaaa gcggaaatat cacatttgag aaagaagttg ctcctctgga aggaacttca 600

gtatgccagg ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660

aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720

gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780

tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840

gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900

ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960

ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020

tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080

ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140

tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200

cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260

ggcaaggtta ttattgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320

attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380

agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440

gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500

aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560

gaaatgaagt cctga 1575

<210> SEQ ID NO: 37

<211> LENGTH: 2142

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ACS1

<400> SEQENCE: 37

atgtcgccct ctgccgtaca atcatcaaaa ctagaagaac agtcaagtga aattgacaag 60

ttgaaagcaa aaatgtccca gtctgccgcc actgcgcagc agaagaagga acatgagtat 120

gaacatttga cttcggtcaa gatcgtgcca caacggccca tctcagatag actgcagccc 180

gcaattgcta cccactattc tccacacttg gacgggttgc aggactatca gcgcttgcac 240

aaggagtcta ttgaagaccc tgctaagttc ttcggttcta aagctaccca atttttaaac 300

tggtctaagc cattcgataa ggtgttcatc ccagacccta aaacgggcag gccctccttc 360

cagaacaatg catggttcct caacggccaa ttaaacgcct gttacaactg tgttgacaga 420

catgccttga agactcctaa caagaaagcc attattttcg aaggtgacga gcctggccaa 480

ggctattcca ttacctacaa ggaactactt gaagaagttt gtcaagtggc acaagtgctg 540

acttactcta tgggcgttcg caagggcgat actgttgccg tgtacatgcc tatggtccca 600

gaagcaatca taaccttgtt ggccatttcc cgtatcggtg ccattcactc cgtagtcttt 660

gccgggtttt cttccaactc cttgagagat cgtatcaacg atggggactc taaagttgtc 720

atcactacag atgaatccaa cagaggtggt aaagtcattg agactaaaag aattgttgat 780

gacgcgctaa gagagacccc aggcgtgaga cacgtcttgg tttatagaaa gaccaacaat 840

ccatctgttg ctttccatgc ccccagagat ttggattggg caacagaaaa gaagaaatac 900

aagacctact atccatgcac acccgttgat tctgaggatc cattattctt gttgtatacg 960

tctggttcta ctggtgcccc caagggtgtt caacattcta ccgcaggtta cttgctggga 1020

gctttgttga ccatgcgcta cacttttgac actcaccaag aagacgtttt cttcacagct 1080

ggagacattg gctggattac aggccacact tatgtggttt atggtccctt actatatggt 1140

tgtgccactt tggtctttga agggactcct gcgtacccaa attactcccg ttattgggat 1200

attattgatg aacacaaagt cacccaattt tatgttgcgc caactgcttt gcgtttgttg 1260

aaaagagctg gtgattccta catcgaaaat cattccttaa aatctttgcg ttgcttgggt 1320

tcggtcggtg agccaattgc tgctgaagtt tgggagtggt actctgaaaa aataggtaaa 1380

aatgaaatcc ccattgtaga cacctactgg caaacagaat ctggttcgca tctggtcacc 1440

ccgctggctg gtggtgttac accaatgaaa ccgggttctg cctcattccc cttcttcggt 1500

attgatgcag ttgttcttga ccctaacact ggtgaagaac ttaacaccag ccacgcagag 1560

ggtgtccttg ccgtcaaagc tgcatggcca tcatttgcaa gaactatttg gaaaaatcat 1620

gataggtatc tagacactta tttgaaccct taccctggct actatttcac tggtgatggt 1680

gctgcaaagg ataaggatgg ttatatctgg attttgggtc gtgtagacga tgtggtgaac 1740

gtctctggtc accgtctgtc taccgctgaa attgaggctg ctattatcga agatccaatt 1800

gtggccgagt gtgctgttgt cggattcaac gatgacttga ctggtcaagc agttgctgca 1860

tttgtggtgt tgaaaaacaa atctagttgg tccaccgcaa cagatgatga attacaagat 1920

atcaagaagc atttggtctt tactgttaga aaagacatcg ggccatttgc cgcaccaaaa 1980

ttgatcattt tagtggatga cttgcccaag acaagatccg gcaaaattat gagacgtatt 2040

ttaagaaaaa tcctagcagg agaaagtgac caactaggcg acgtttctac attgtcaaac 2100

cctggcattg ttagacatct aattgattcg gtcaagttgt aa 2142

<210> SEQ ID NO: 38

<211> LENGTH: 1395

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized pduP

<400> SEQENCE: 38

atgaatactt ctgaactcga aaccctgatt cgcaccattc ttagcgagca attaaccacg 60

ccggcgcaaa cgccggtcca gcctcagggc aaagggattt tccagtccgt gagcgaggcc 120

atcgacgccg cgcaccaggc gttcttacgt tatcagcagt gcccgctaaa aacccgcagc 180

gccattatca gcgcgatgcg tcaggagctg acgccgctgc tggcgcccct ggcggaagag 240

agcgccaatg aaacggggat gggcaacaaa gaagataaat ttctcaaaaa caaggctgcg 300

ctggacaaca cgccgggcgt agaagatctc accaccaccg cgctgaccgg cgacggcggc 360

atggtgctgt ttgaatactc accgtttggc gttatcggtt cggtcgcccc aagcaccaac 420

ccgacggaaa ccatcatcaa caacagtatc agcatgctgg cggcgggcaa cagtatctac 480

tttagcccgc atccgggagc gaaaaaggtc tctctgaagc tgattagcct gattgaagag 540

attgccttcc gctgctgcgg catccgcaat ctggtggtga ccgtggcgga acccaccttc 600

gaagcgaccc agcagatgat ggcccacccg cgaatcgcag tactggccat taccggcggc 660

ccgggcattg tggcaatggg catgaagagc ggtaagaagg tgattggcgc tggcgcgggt 720

aacccgccct gcatcgttga tgaaacggcg gacctggtga aagcggcgga agatatcatc 780

aacggcgcgt cattcgatta caacctgccc tgcattgccg agaagagcct gatcgtagtg 840

gagagtgtcg ccgaacgtct ggtgcagcaa atgcaaacct tcggcgcgct gctgttaagc 900

cctgccgata ccgacaaact ccgcgccgtc tgcctgcctg aaggccaggc gaataaaaaa 960

ctggtcggca agagcccatc ggccatgctg gaagccgccg ggatcgctgt ccctgcaaaa 1020

gcgccgcgtc tgctgattgc gctggttaac gctgacgatc cgtgggtcac cagcgaacag 1080

ttgatgccga tgctgccagt ggtaaaagtc agcgatttcg atagcgcgct ggcgctggcc 1140

ctgaaggttg aagaggggct gcatcatacc gccattatgc actcgcagaa cgtgtcacgc 1200

ctgaacctcg cggcccgcac gctgcaaacc tcgatattcg tcaaaaacgg cccctcttat 1260

gccgggatcg gcgtcggcgg cgaaggcttt accaccttca ctatcgccac accaaccggt 1320

gaagggacca cgtcagcgcg tacttttgcc cgttcccggc gctgcgtact gaccaacggc 1380

ttttctattc gctaa 1395

<210> SEQ ID NO: 39

<211> LENGTH: 1149

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized fucO

<400> SEQENCE: 39

atggctaaca gaatgattct gaacgaaacg gcatggtttg gtcggggtgc tgttggggct 60

ttaaccgatg aggtgaaacg ccgtggttat cagaaggcgc tgatcgtcac cgataaaacg 120

ctggtgcaat gcggcgtggt ggcgaaagtg accgataaga tggatgctgc agggctggca 180

tgggcgattt acgacggcgt agtgcccaac ccaacaatta ctgtcgtcaa agaagggctc 240

ggtgtattcc agaatagcgg cgcggattac ctgatcgcta ttggtggtgg ttctccacag 300

gatacttgta aagcgattgg cattatcagc aacaacccgg agtttgccga tgtgcgtagc 360

ctggaagggc tttccccgac caataaaccc agtgtaccga ttctggcaat tcctaccaca 420

gcaggtactg cggcagaagt gaccattaac tacgtgatca ctgacgaaga gaaacggcgc 480

aagtttgttt gcgttgatcc gcatgatatc ccgcaggtgg cgtttattga cgctgacatg 540

atggatggta tgcctccagc gctgaaagct gcgacgggtg tcgatgcgct cactcatgct 600

attgaggggt atattacccg tggcgcgtgg gcgctaaccg atgcactgca cattaaagcg 660

attgaaatca ttgctggggc gctgcgagga tcggttgctg gtgataagga tgccggagaa 720

gaaatggcgc tcgggcagta tgttgcgggt atgggcttct cgaatgttgg gttagggttg 780

gtgcatggta tggcgcatcc actgggcgcg ttttataaca ctccacacgg tgttgcgaac 840

gccatcctgt taccgcatgt catgcgttat aacgctgact ttaccggtga gaagtaccgc 900

gatatcgcgc gcgttatggg cgtgaaagtg gaaggtatga gcctggaaga ggcgcgtaat 960

gccgctgttg aagcggtgtt tgctctcaac cgtgatgtcg gtattccgcc acatttgcgt 1020

gatgttggtg tacgcaagga agacattccg gcactggcgc aggcggcact ggatgatgtt 1080

tgtaccggtg gcaacccgcg tgaagcaacg cttgaggata ttgtagagct ttaccatacc 1140

gcctggtaa 1149

<210> SEQ ID NO: 40

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ATOA_ECOLI catalytic GLU location

<400> SEQENCE: 40

Ile Thr Leu Gln Ser Glu Asn Gly Phe Leu

1 5 10

<210> SEQ ID NO: 41

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized ITLQSENGFL catalytic GLU location

<400> SEQENCE: 41

Ile Thr Leu Gln Ser Glu Asn Gly Phe Leu

1 5 10

<210> SEQ ID NO: 42

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized CATJ_PSESB catalytic GLU location

<400> SEQENCE: 42

Val Val Leu Ile Tyr Glu Ser Gly Pro Ile

1 5 10

<210> SEQ ID NO: 43

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized CTFB_CLOAB catalytic GLU location

<400> SEQENCE: 43

Ile Thr Phe Gln Ser Glu Asn Gly Ile Val

1 5 10

<210> SEQ ID NO: 44

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized GCTB_ACIFV catalytic GLU location

<400> SEQENCE: 44

Cys His Ile Ile Val Glu Ser Gly Leu Met

1 5 10

<210> SEQ ID NO: 45

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PCAJ_ACIAD catalytic GLU location

<400> SEQENCE: 45

Val Phe Leu His Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 46

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PCAJ_PSEPK catalytic GLU location

<400> SEQENCE: 46

Val Phe Leu His Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 47

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized PCAJ_PSEPU catalytic GLU location

<400> SEQENCE: 47

Val Phe Leu His Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 48

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_BACSU catalytic GLU location

<400> SEQENCE: 48

Val Met Leu Gln Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 49

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_HELPJ catalytic GLU location

<400> SEQENCE: 49

Ile Val Phe Gln Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 50

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_HELPY catalytic GLU location

<400> SEQENCE: 50

Ile Val Phe Gln Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 51

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_MYCBO catalytic GLU location

<400> SEQENCE: 51

Val Val Leu His Ser Glu Asn Gly Ile Leu

1 5 10

<210> SEQ ID NO: 52

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_MYCTU catalytic GLU location

<400> SEQENCE: 52

Val Val Leu His Ser Glu Asn Gly Ile Leu

1 5 10

<210> SEQ ID NO: 53

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_XANCB catalytic GLU location

<400> SEQENCE: 53

Val Trp Leu Gln Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 54

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized SCOB_XANCP catalytic GLU location

<400> SEQENCE: 54

Val Trp Leu Gln Ser Glu Asn Gly Leu Leu

1 5 10

<210> SEQ ID NO: 55

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Y3552_MYCTU catalytic GLU location

<400> SEQENCE: 55

Ile Leu Leu Thr Asp Gly Glu Ala Gln Leu

1 5 10

<210> SEQ ID NO: 56

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized Y3582_MYCBO catalytic GLU location

<400> SEQENCE: 56

Ile Leu Leu Thr Asp Gly Glu Ala Gln Leu

1 5 10

<210> SEQ ID NO: 57

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized YODR BACSU catalytic GLU location

<400> SEQENCE: 57

Val Met Phe Gln Ala Glu Asn Gly Val Leu

1 5 10

<210> SEQ ID NO: 58

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthesized YDIF ECOLI catalytic GLU location

<400> SEQENCE: 58

Phe Ile Leu Thr Val Glu Thr Gly Pro Ile

1 5 10

Read more
PatSnap Solutions

Great research starts with great data.

Use the most comprehensive innovation intelligence platform to maximise ROI on research.

Learn More

Patent Valuation

$

Reveal the value <>

24.28/100 Score

Market Attractiveness

It shows from an IP point of view how many competitors are active and innovations are made in the different technical fields of the company. On a company level, the market attractiveness is often also an indicator of how diversified a company is. Here we look into the commercial relevance of the market.

37.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

72.41/100 Score

Technology Quality

It shows the degree of innovation that can be derived from a company’s IP. Here we look into ease of detection, ability to design around and significance of the patented feature to the product/service.

50.0/100 Score

Assignee Score

It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

19.79/100 Score

Legal Score

It shows the legal strength of IP in terms of its degree of protecting effect. Here we look into claim scope, claim breadth, claim quality, stability and priority.

Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
Process for production of isopropanol, and genetically modified yeast capable of producing isopropanol TOYOTA JIDOSHA KABUSHIKI KAISHA 14 May 2010 20 March 2013
Methods and compositions for the recombinant biosynthesis of propanol JOULE UNLIMITED TECHNOLOGIES, INC.,SKRALY, FRANK, ANTHONY 13 May 2011 17 November 2011
Modified microorganisms and methods of making butadiene using same BRASKEM S.A.,GARCEZ LOPES, MATEUS SCHREINER,SLOVIC, AVRAM MICHAEL,GOUVEA, IURI ESTRADA,PEREZ, JOHANA RINCONES 17 December 2012 20 June 2013
Modified microorganism and methods of using same for producing 2- propanol and 1-propanol and/or 1.2-propanediol BRASKEM S.A.,SLOVIC, AVRAM MICHAEL 05 September 2014 12 March 2015
Fermentative production of acetone from renewable resources by means of novel metabolic pathway EVONIK DEGUSSA GMBH 01 October 2008 14 October 2010
See full citation <>

More like this

Title Current Assignee Application Date Publication Date
Microorganisms and methods for the co-production of ethylene glycol and three carbon compounds BRASKEM S.A. 08 March 2017 14 September 2017
Production of propanol and/or propionic acid EVONIK DEGUSSA GMBH 28 July 2016 16 February 2017
Method for producing 3-hydroxypropionic acid and other products OPX BIOTECHNOLOGIES, INC.,THE REGENTS OF THE UNIVERSITY OF COLORADO,LYNCH, MICHAEL, D.,GILL, RYAN, T.,WARNECKE-LIPSCOMB, TANYA 27 September 2010 31 March 2011
Synthesis of omega functionalized methylketones, 2-alcohols, 2-amines, and derivatives thereof WILLIAM MARSH RICE UNIVERSITY 15 April 2016 20 October 2016
Enzymatic methods for butanol production NEWPEK S.A. DE C.V. 05 May 2016 09 November 2017
Process for the biological production of 1,3-propanediol with high titer E. I. DU PONT DE NEMOURS AND COMPANY 16 January 2006 17 March 2009
Production of 3-hydroxybutyrate EVONIK DEGUSSA GMBH 15 July 2016 02 February 2017
Recombinant microorganisms and uses therefor LANZATECH NEW ZEALAND LIMITED 23 February 2012 09 August 2016
Modified photosynthetic microorganisms for producing lipids MATRIX GENETICS, LLC,ROBERTS, JAMES,CROSS, FRED,MCCORMICK, MARGARET MARY,MUNOZ, ERNESTO JAVIER 19 December 2011 28 June 2012
Method for the production of isoamyl alcohol GLOBAL BIOENERGIES 23 June 2016 29 December 2016
Anoxic biological production of fuels and of bulk chemicals from second generation feedstocks ABENGOA BIOENERGIA NUEVAS TECNOLOGIAS S.A. 26 June 2014 31 December 2014
Improved production of itaconic acid LESAFFRE ET COMPAGNIE 25 August 2017 01 March 2018
Transformant of coryneform bacteria capable of producing isopropanol RESEARCH INSTITUTE OF INNOVATIVE TECHNOLOGY FOR THE EARTH 15 April 2009 10 July 2012
A microorganism modified for the production of 1,3-propanediol INSTITUT NATIONAL DES SCIENCES APPLIQUÉES 10 July 2013 16 January 2014
Microorganism engineered to produce isopropanol THE REGENTS OF THE UNIVERSITY OF CALIFORNIA,LIAO, JAMES, C.,ATSUMI, SHOTA,HANAI, TAIZO 11 October 2008 28 May 2009
New lactaldehyde reductases for the production of 1,2-propanediol METABOLIC EXPLORER 10 September 2015 16 March 2017
Acetyl-COA producing enzymes in yeast DSM IP ASSETS B.V.,MUELLER, ULRIKE, MARIA,WU, LIANG,RAAMSDONK, LOURINA, MADELEINE,WINKLER, AARON ADRIAAN 11 July 2008 29 January 2009
Microbial production of 1,2-propanediol from sugar WISCONSIN ALUMNI RESEARCH FOUNDATION 07 February 2000 16 October 2001
Genetically modified microorganism and production method for aliphatic polyester using same TOYOTA JIDOSHA KABUSHIKI KAISHA,MURAMATSU MASAYOSHI,KAMBE HIROMI,ITO MASAKAZU,SHIMAMURA TAKASHI 24 March 2011 29 September 2011
Method for industrial manufacture of chiral-1,1-difluoro-2-propanol PUBLIC UNIVERSITY CORPORATION TOYAMA PREFECTURAL UNIVERSITY,CENTRAL GLASS COMPANY, LIMITED 02 March 2016 09 September 2016
See all similar patents <>

More Patents & Intellectual Property

PatSnap Solutions

PatSnap solutions are used by R&D teams, legal and IP professionals, those in business intelligence and strategic planning roles and by research staff at academic institutions globally.

PatSnap Solutions
Search & Analyze
The widest range of IP search tools makes getting the right answers and asking the right questions easier than ever. One click analysis extracts meaningful information on competitors and technology trends from IP data.
Business Intelligence
Gain powerful insights into future technology changes, market shifts and competitor strategies.
Workflow
Manage IP-related processes across multiple teams and departments with integrated collaboration and workflow tools.
Contact Sales
Clsoe
US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 1 US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 2 US10000744 Engineered enzyme acetoacetyl-CoA hydrolase 3