KR102598992B1 - Isobutanol and polyhydroxybutyrate simultaneous production strain and use thereof - Google Patents

Abstract

The present invention relates to strains that simultaneously produce isobutanol and polyhydroxybutyrate (PHB) and their use.
The strain for simultaneous production of isobutanol and polyhydroxybutyrate (PHB) according to the present invention is economical by simultaneously utilizing culture medium and cell mass, exhibits increased cell activity and faster glucose utilization, and robustness of the strain. This is improved, can reduce acetate accumulation during fermentation without gene deletion, and does not require additional processes to separate the final product, so it can be applied to the industrial production of isobutanol and PHB.

Description

Isobutanol and polyhydroxybutyrate simultaneous production strain and use thereof {Isobutanol and polyhydroxybutyrate simultaneous production strain and use thereof}

The present invention relates to strains that simultaneously produce isobutanol and polyhydroxybutyrate (PHB) and their use.

Isobutanol is a branched-chain aliphatic C4 alcohol, commonly used as a gasoline blending material and as a precursor in the synthesis of butyl rubber, lubricants, and polyester. This chemical is considered one of the most potent hydrocarbon fuels because it has a higher energy capacity than ethanol and is less miscible with water, causing less corrosion in pipelines and fuel delivery infrastructure. The synthetic isobutanol production operon is Escherichia coli , E. coli , Saccharomyces cerevisiae , S. cerevisiae , Corynebacterium glutamicum , C. glutamicum , and Ralstonia. Eutropha ( Ralstonia eutropha , R. eutropha ), Bacillus subtilis ( B. subtilis ), Pseudomonas putida ( P. putida ), and Zymomonas mobilis ( Z. mobilis ), etc. There is.

Several approaches have been attempted to optimize the microbial process for isobutanol production. For fermentation, an in situ removal approach was applied to collect isobutanol using its volatility. Because cell growth is hindered by alcohol concentrations above 8 g/L, transcriptional and evolutionary studies have been performed to increase tolerance to isobutanol in E. coli , manipulating NAD(P)H levels in cells; Modifying amino acid residues of α-ketoisovalerate decarboxylase using protein engineering; Integration of expression of multiple genes to utilize carbon sources other than glucose, such as cellobiose, protein waste, or acetate; Achieving flux balance for optimal production; Several metabolic engineering studies have been performed, including:

Meanwhile, polyhydroxybutyrate (PHB) is a carbon storage biopolymer that accumulates intracellularly at concentrations of up to 80% in various types of microorganisms such as E. coli and R. eutropha . PHB has a wide range of potential applications, including use as an implant material and container production, due to its advantageous mechanical, biodegradability and histocompatibility properties when appropriately modified with different monomers. Despite these valuable properties, the use of PHB has been limited at the industrial level due to its high production costs.

Accordingly, many researchers have focused on PHB production using low-cost substrates. Since the polymers are stored in cells, co-production strategies have been investigated to investigate the possibility of producing other valuable extracellular chemicals without additional separation processes to achieve an economic balance. In a previous study, it was confirmed that using PHB as a storage biochemical in a fermentation process involving other microorganisms had a positive effect on the co-production strategy (Li, T., et al. 2017. Co-production of microbial polyhydroxyalkanoates with other chemicals. Metab. Eng. 43, 29-36.).

However, biofuel and biopolymer production by microorganisms still has serious and persistent environmental problems and causes global warming. Additionally, many obstacles remain to achieve optimal bioindustrial processes for biofuel and biopolymer production, including the use of leftover biomass, problems associated with final product separation, and production costs.

Against this background, the present inventors selected Escherichia coli as a model organism and metabolically engineered it to produce PHB and isobutanol simultaneously, resulting in economical, increased cell activity and faster glucose utilization by simultaneously utilizing culture medium and cell mass. By developing a strain that exhibits improved strain robustness, can reduce acetate accumulation during fermentation without gene deletion, and does not require additional processes to isolate the final product, it can be applied to the industrial production of isobutanol and PHB. The present invention was completed by confirming that it is possible.

The purpose of the present invention is to provide a strain that simultaneously produces isobutanol and polyhydroxybutyrate, expressing the isobutanol synthesis operon, the polyhydroxybutyrate (PHB) synthesis operon, and the pntAB gene.

Another object of the present invention is i) culturing the strain in a medium; and ii) recovering isobutanol and polyhydroxybutyrate from the cultured medium and strain.

Another object of the present invention is to provide a composition for simultaneous production of isobutanol and polyhydroxybutyrate, comprising the above strain and its culture medium.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention for achieving the above object is to produce a strain simultaneously producing isobutanol and polyhydroxybutyrate, which expresses the isobutanol synthesis operon, the polyhydroxybutyrate (PHB) synthesis operon, and the pntAB gene. to provide.

For the purpose of the present invention, the isobutanol synthesis operon may be any one or more selected from the group consisting of alsS , kivD , ilvC , ilvD genes and combinations thereof, for example, including alsS , kivD , ilvC and ilvD genes. It may be an operon. The PHB synthesis operon may be any one or more selected from the group consisting of bktB , phaB , phaC genes, and combinations thereof. For example, it may be an operon containing bktB , phaB , and phaC genes.

That is, as an example of the strain of the present invention, the strain may express alsS , kivD , ilvC , ilvD , pntAB , bktB , phaB , and phaC genes, but is not limited thereto.

In the present invention, the term acetolactate synthase is an enzyme that catalyzes the first step in the synthesis of branched chain amino acids. Specifically, the acetolactate synthase of the present invention can be used interchangeably with acetolactate synthase or alsS. In the present invention, the sequence of the acetolactate synthase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide having acetolactate synthase activity encoded by alsS , but is not limited thereto.

In the present invention, the term alpha-ketoisovalerate decarboxylase is a non-oxidizing enzyme that exhibits the highest affinity for alpha-ketoisovalerate, an intermediate compound in valine and leucine biosynthesis. It is a thiamine diphosphate (non-oxidative thiamin diphosphate, ThDP) dependent alpha-keto acid decarboxylase. Specifically, alpha-ketoisovalerate decarboxylase of the present invention can be used interchangeably with alpha-ketoisovalerate decarboxylase or kivD. In the present invention, the sequence of alpha-ketoisovalerate decarboxylase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide having alpha-ketoisovalerate decarboxylase activity encoded by kivD , but is not limited thereto.

In the present invention, the term ketol-acid reductoisomerase refers to (R)-2,3-dihydroxy-3-methylbutyrate [(R)-2,3-dihydroxy-3-methylbutanoate] and (S)-2-hydroxy-2-methyl-3-oxobutanoate [(S)-2-hydroxy-2-methyl-3-oxobutanoate], Nicotinamide-adenine dinucleotide phosphate (NADPH), and H+ from NADP+. It is an enzyme that produces Specifically, ketol acid reductoisomerase of the present invention can be used interchangeably with ilvC. In the present invention, the sequence of the ketol acid reductoisomerase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide having ketol acid reductoisomerase activity encoded by ilvC , but is not limited thereto.

In the present invention, the term dihydroxyacid dehydratase is an enzyme that catalyzes a key step in the branched chain amino acid biosynthesis pathway. Specifically, the dihydroxy acid dehydratase of the present invention can be used interchangeably with dihydroxy acid dehydratase or ilvD. In the present invention, the sequence of the acetolactate synthase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide having dihydroxy acid dehydratase activity encoded by ilvD , but is not limited thereto.

In the present invention, the term pyridine nucleotide transhydrogenase is a catalyst that catalyzes the first step in the synthesis of branched chain amino acids. Specifically, pyridine nucleotide transhydrogenase of the present invention can be used interchangeably with pyridine nucleotide transhydrogenase or pntAB. In the present invention, the sequence of the pyridine nucleotide trans hydrogenase can be obtained from NCBI's GenBank, a known database. Specifically, it may be a protein or polypeptide having pyridine nucleotide trans hydrogenase activity encoded by pntAB , but is not limited thereto.

In the present invention, the term beta-ketothiolase decomposes ketone acetoacetyl CoA into acetyl-CoA and produces 2-methylacetoacetyl-CoA in the isoleucine catabolism pathway. It is an enzyme that converts CoA) into propionic acid. Specifically, beta-ketothiolase of the present invention can be used interchangeably with mitochondrial acetoacetyl-CoA thiolase (MAT) or bktB. In the present invention, the sequence of the beta-ketothiolase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide with beta-ketothiolase activity encoded by bktB , but is not limited thereto.

In the present invention, the term acetoacetyl-CoA reductase (acetoacetyl-CoA reductase) refers to 3-oxoacyl-CoA (from (R)-3-hydroxyacyl-CoA [(R)-3-hydroxyacyl-CoA] and NADP + 3-oxoacyl-CoA), an enzyme that produces NADPH and H+. Specifically, acetoacetyl-CoA reductase of the present invention can be used interchangeably with acetoacetyl-CoA reductase or phaB. In the present invention, the sequence of the acetoacetyl-CoA reductase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide having acetoacetyl-CoA reductase activity encoded by phaB , but is not limited thereto.

In the present invention, the term poly(3-hydroxybutyrate) polymerase is an enzyme that has the activity of transferring an acyl group. Specifically, poly(3-hydroxybutyrate)polymerase of the present invention can be used interchangeably with poly(3-hydroxybutyrate)polymerase or phaC. In the present invention, the sequence of the poly(3-hydroxybutyrate)polymerase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a protein or polypeptide with poly(3-hydroxybutyrate)polymerase activity encoded by phaC , but is not limited thereto.

In the present invention, each protein (or polypeptide) of alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB and phaC is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, It may include the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. Specifically, of the present invention, alsS is the amino acid sequence of SEQ ID NO: 2, kivD is the amino acid sequence of SEQ ID NO: 4, ilvC is the amino acid sequence of SEQ ID NO: 6, ilvD is the amino acid sequence of SEQ ID NO: 8, and pntAB is the amino acid sequence of SEQ ID NO: 10. , bktB may have, include, or consist of the amino acid sequence of SEQ ID NO: 12, phaB may have the amino acid sequence of SEQ ID NO: 14, and phaC may have, include, or consist of the amino acid sequence of SEQ ID NO: 16, respectively, or may consist essentially of the above amino acid sequences.

In the present invention, the term "polynucleotide" refers to a strand of DNA or RNA of a certain length or more, which is a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds. More specifically, the term "polynucleotide" refers to a strand of DNA or RNA that encodes the protein. refers to a polynucleotide fragment.

Polynucleotides encoding alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB and phaC of the present invention are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: It may include a base sequence encoding the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16.

As an example of the present invention, each gene encoding the proteins alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB and phaC, that is, alsS , kivD , ilvC , ilvD , pntAB , bktB , phaB and phaC genes are each It may include a polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. Specifically, the gene encoding alsS of the present invention is the polynucleotide sequence of SEQ ID NO: 1, the gene encoding kivD is the polynucleotide sequence of SEQ ID NO: 3, and the gene encoding ilvC is the polynucleotide sequence of SEQ ID NO: 5, ilvD. The coding gene is the polynucleotide sequence of SEQ ID NO: 7, the gene encoding pntAB is the polynucleotide sequence of SEQ ID NO: 9, the gene encoding bktB is the polynucleotide sequence of SEQ ID NO: 11, and the gene encoding phaB is the polynucleotide sequence of SEQ ID NO: 13. The polynucleotide sequence, the gene encoding phaC, may have, include, or consist of the polynucleotide sequence of SEQ ID NO: 15, or may consist essentially of the polynucleotide sequence.

The polynucleotide of the present invention has various variations in the coding region within the range of not changing the amino acid sequence of the protein of the present invention, taking into account codon degeneracy or preferred codons in organisms intended to express the protein of the present invention. Transformations can occur. Specifically, the polynucleotide of the present invention has 70% homology or identity with the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. or more than 75%, more than 6%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, and more than 99% of the base sequence, or SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 with at least 70%, 75%, 6%, or 85% homology or identity. It may consist of, or consist essentially of, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, and more than 99% of base sequences, but is not limited thereto.

In addition, the polynucleotide of the present invention is without limitation as long as it is a probe that can be prepared from a known genetic sequence, for example, a sequence capable of hydriding under strict conditions with a complementary sequence to all or part of the polynucleotide sequence of the present invention. may be included. The “stringent condition” refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, among polynucleotides with high homology or identity, 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, Or, conditions in which polynucleotides with 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or 60°C, which is the washing condition for normal southern hybridization, Washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, more specifically 68°C, 0.1ХSSC, 0.1% SDS. Conditions can be listed.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases may be possible depending on the stringency of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, polynucleotides of the invention may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

Specifically, polynucleotides having homology or identity with the polynucleotide of the present invention can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art depending on the purpose.

The appropriate stringency to hybridize the polynucleotide depends on the length of the polynucleotide and the degree of complementarity, variables that are well known in the art (e.g., J. Sambrook et al., supra).

In addition, alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB and phaC of the present invention are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or At least 70%, 75%, 6%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology with the amino acid sequence shown in SEQ ID NO: 16 Alternatively, it may contain an amino acid sequence having identity. In addition, proteins with amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present invention, as long as they have such homology or identity and show efficacy corresponding to the protein of the present invention. is self-explanatory.

For example, addition or deletion of sequences at the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the protein of the present invention, mutations that may occur naturally, silent mutations or preservation. This is the case with enemy substitution.

The term “conservative substitution” means replacing one amino acid with another amino acid having similar structural and/or chemical properties. These amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In the present invention, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may be used with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of a sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with a polynucleotide containing a common codon or a codon taking codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: It can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Or, as performed in the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST from the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity or identity of polynucleotides or polypeptides is defined in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. This can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

Any one or more proteins selected from the group consisting of alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB, phaC and combinations thereof expressed by the strain of the present invention are alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB Alternatively, it may be introduced through a vector containing a polynucleotide encoding phaC, that is, alsS , kivD , ilvC , ilvD , pntAB , bktB , phaB , and phaC genes.

The vector of the present invention may include a DNA preparation containing the base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) to enable expression of the target polypeptide in a suitable host. You can. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used, and as plasmid vectors, RSF, P15A, and pDZ series can be used. , pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Specifically, pRSFDuet-1, pACYCDuet-1, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. The selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of the target nucleic acid molecule, and to display selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that provide may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

In the present invention, the term “transformation” refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoding the polynucleotide can be expressed within the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it can include both of these, regardless of whether it is inserted into the chromosome of the host cell or located outside the chromosome. Additionally, the polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target protein of the present invention.

The strain of the present invention may contain a protein of the present invention, a polynucleotide encoding the protein, or a vector containing the polynucleotide of the present invention.

In the present invention, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and can be caused by insertion of foreign genes or enhanced or inactivated activity of intrinsic genes. It is a microorganism whose specific mechanism is weakened or strengthened, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product.

The strain of the present invention is a strain containing any one or more of the protein of the present invention, the polynucleotide of the present invention, and the vector containing the polynucleotide of the present invention; Strains modified to express proteins of the invention or polynucleotides of the invention; A strain (e.g., a recombinant strain) expressing a protein of the invention, or a polynucleotide of the invention; Alternatively, it may be a strain (e.g., a recombinant strain) having the protein activity of the present invention, but is not limited thereto.

The strain of the present invention may be a strain capable of producing isobutanol and PHB simultaneously.

The strain of the present invention is a microorganism that naturally has alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB or phaC or has the ability to produce isobutanol or PHB, or alsS, kivD, ilvC, ilvD, pntAB, bktB, phaB Or, the protein of the present invention or a polynucleotide (or a vector containing the polynucleotide) encoding the same is introduced into a parent strain that does not have phaC or is incapable of producing isobutanol or PHB, and/or is given the ability to simultaneously produce isobutanol and PHB. It may be a microorganism, but is not limited thereto.

As an example, the strain of the present invention is a cell or microorganism that is transformed with a vector containing a polynucleotide encoding the polynucleotide of the present invention or a polynucleotide encoding the protein of the present invention and expresses the protein of the present invention. The strain of the invention may include all microorganisms capable of simultaneously producing isobutanol and PHB, including the protein of the invention. For example, the strain of the present invention expresses the protein by introducing a polynucleotide encoding the protein of the present invention into a natural wild-type microorganism or a microorganism that produces isobutanol or PHB, and is given the ability to simultaneously produce isobutanol and PHB. It may be a recombinant strain. The recombinant strain endowed with the ability to simultaneously produce isobutanol and PHB may be a strain that simultaneously produces isobutanol and PHB compared to a natural wild-type microorganism or an unmodified microorganism (i.e., a microorganism that does not express the protein of the present invention). It is not limited to this. An example of this may be the BLISO strain containing the vector pRSFISO containing the isobutanol synthesis operon, which is the target strain for comparing the simultaneous production of isobutanol and PHB, but is not limited thereto.

In the present invention, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and is either a wild-type strain or a natural strain itself, or a strain that has a genetic mutation due to natural or artificial factors. It may refer to the strain before change. For example, the unmodified microorganism may refer to a strain in which the protein described herein is not introduced or before the protein is introduced. The “non-modified microorganism” may be used interchangeably with “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated strain”, “non-modified strain”, “non-mutated microorganism” or “reference microorganism”.

As another example of the present invention, the microorganism of the present invention is not particularly limited as long as it is a microorganism capable of simultaneously producing isobutanol and PHB, but may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. The microorganisms in the Corynebacterium genus are specifically, Corynebacterium glutamicum , Corynebacterium ammoniagenes , Brevibacterium lactofermentum , and Brevibacter. It may be Brevibacterium flavum , Corynebacterium thermoaminogenes , Corynebacterium efficiens , etc.

The microorganisms of the Escherichia genus are specifically, Escherichia albertii , Escherichia coli, Escherichia fergusonii , Escherichia hermannii , S. It may be Escherichia vulneris , etc.

More specifically, the microorganism of the present invention may be Escherichia coli, but is not limited thereto.

Modification of part or all of the polynucleotide in the microorganism of the present invention is (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by, but is not limited to, light and/or chemical treatment, such as ultraviolet rays and radiation. The method of modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the gene of interest is injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

In one embodiment of the present invention, a strain producing isobutanol and PHB simultaneously was produced by introducing the PHB synthesis operon ( bktB , phaB , phaC ) into a strain containing the isobutanol synthesis operon ( alsS , kivD , ilvC , ilvD ). , cell growth was activated by the PHB synthesis operon (Figure 1A), and isobutanol production was increased approximately 1.6-fold (1.14 g/L) in the engineered E. coli (Figure 1B).

Additionally, the isobutanol productivity conferred to E. coli by the PHB synthesis operon is generally proportional to cell growth, and the PHB synthesis operon was shown to activate cell activity (Figure 3).

The above results confirmed that expression of the PHB synthetic operon improved isobutanol production.

Additionally, the distribution of cofactors in biosynthetic pathways is important for balancing biomass accumulation and metabolite production, and there are many pathways involved in maintaining redox balance, including the electron transport chain and the tricarboxylic acid (TCA) cycle. IlvC and ADH in the isobutanol pathway and PhaB in the PHB synthesis pathway are known to utilize NAD(P)H as a cofactor for each reaction. Therefore, overexpression of both the isobutanol and PHB synthesis operons may lead to redox imbalance, which in turn may limit productivity, and genes to be overexpressed were selected as the driving force for cofactor formation.

In this regard, in one embodiment of the present invention, pntAB was analyzed to play an active role in promoting cell growth and isobutanol (2.7 g/L) and PHB synthesis (0.52 g/L), resulting in isobutanol and PHB co-production. It was confirmed to be suitable for production (Figure 4), and was expressed in the strain together with the isobutanol synthesis operon and the PHB synthesis operon.

Meanwhile, in one embodiment of the present invention, the isobutanol and PHB co-producing strain (BLpLISOpntAB) of the present invention showed faster cell growth and higher yield (Figure 6A). Isobutanol and PHB production were approximately 2.4 g/L and 0.25 g/L, respectively, which were two times higher than that of the BLISO strain containing only the vector pRSFISO (Figure 6B). Moreover, glucose consumption was slightly faster and more complete, and acetate accumulation was maintained below 0.5 g/L in cultures of the BLpLISOpntAB strain (Figure 6C,D).

These results indicated that the isobutanol and PHB co-producing strains were robust. The PHB synthesis pathway uses acetyl-CoA for PHB production, which prevented acetate overflow when glucose was the only carbon source.

Moreover, maintaining low acetate concentrations in the culture medium increased the utilization of glucose as evidenced by glucose consumption. As a result, it was confirmed that the high metabolic activity and cell growth of E. coli are advantageous for the co-production of isobutanol and PHB.

In addition, solvent toxicity tests were performed including ethanol (40 g/L), isopropanol (20 g/L), isobutanol (10 g/L), and n-butanol (10 g/L), which can promote osmotic shock of the strain. As a result, cell growth was increased by the PHB module system in relation to the amount of biomass at the end of fermentation, thus confirming that PHB synthesis can increase the resistance of engineered E. coli to isobutanol stress.

That is, it was confirmed that the isobutanol and PHB co-producing strain according to the present invention exhibited increased cell activity, faster glucose utilization, and lower acetate accumulation in the culture medium.

Another aspect of the present invention includes i) culturing the strain of the present invention in a medium; and ii) recovering isobutanol and polyhydroxybutyrate from the cultured medium and strain; providing a method for simultaneous production of isobutanol and polyhydroxybutyrate (PHB).

In the present invention, the term “culture” means growing the strain of the present invention under appropriately controlled environmental conditions. The culture process of the present invention can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

In the present invention, the term "medium" refers to a material that is mainly mixed with nutrients necessary for cultivating the strain of the present invention, and supplies nutrients and growth factors, including water, which are essential for survival and growth. . Specifically, the medium and other culture conditions used for cultivating the strain of the present invention can be any medium used for cultivating ordinary microorganisms without particular limitation, but the strain of the present invention can be grown with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins, while controlling temperature, pH, etc.

In the present invention, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used individually or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and/or appropriate precursors. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

During cultivation of the strain of the present invention, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited to this. That is not the case.

Additionally, the medium of the present invention may contain yeast extract at 0.1%v/v to 1%v/v, specifically 0.1%v/v to 0.6%v/v, more specifically 0.3%v/v. It can be included.

Because the yeast extract is rich in nutrients, in one embodiment of the present invention, cell growth and isobutanol titer were proportional to the yeast extract concentration added to the medium (FIG. 4A and B). 0.3 %v/v yeast extract produced 1.4 and 0.2 g/L of isobutanol and PHB, respectively, but these levels gradually decreased when the yeast extract concentration was higher than 0.3 %v/v (Figure 4B). Therefore, it was confirmed that the optimal yeast extract concentration for co-production of isobutanol and PHB is 0.1%v/v to 0.6%v/v, specifically 0.3%v/v.

In the culture of the present invention, the culture temperature can be maintained at 20 to 40°C, specifically 25 to 37°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

Isobutanol produced by the culture of the present invention is secreted into the medium, and PHB may remain in the cells.

The isobutanol and PHB production method of the present invention includes preparing the strain of the present invention, preparing a medium for culturing the strain, or a combination thereof (in any order), for example , may be additionally included before the culturing step.

The method can recover isobutanol and PHB from the cultured medium or strain.

The recovery may be to collect the desired IMP using a suitable method known in the art according to the method of cultivating the microorganism of the present invention, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatographies such as chromatography, HPLC, or a combination of these methods can be used, and the desired isobutanol and PHB can be recovered from the medium or microorganisms using suitable methods known in the art.

Specifically, isobutanol can be recovered from the medium and polyhydroxybutyrate can be recovered from the strain.

In one embodiment of the present invention, PHB and isobutanol were found in different locations (intracellular and extracellular, respectively) during the fermentation process (Figure 2).

In other words, it was confirmed that the isobutanol and PHB co-producing strain according to the present invention does not require an additional process to separate PHB and isobutanol when fermentation is completed.

Additionally, the isobutanol and PHB production method of the present invention may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the method for producing isobutanol and PHB of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously in any order, simultaneously or in one step. It may be performed in an integrated manner, but is not limited thereto.

Another aspect of the present invention provides a composition for simultaneous production of isobutanol and polyhydroxybutyrate (PHB), comprising the strain of the present invention and its culture medium.

The terms used here are the same as described above.

The composition includes the strain of the present invention and its culture medium, and may further include without limitation components that can increase the simultaneous production of isobutanol and PHB by the strain.

The strain for simultaneous production of isobutanol and polyhydroxybutyrate (PHB) according to the present invention is economical by simultaneously utilizing culture medium and cell mass, exhibits increased cell activity and faster glucose utilization, and robustness of the strain. This is improved, can reduce acetate accumulation during fermentation without gene deletion, and does not require additional processes to separate the final product, so it can be applied to the industrial production of isobutanol and PHB.

Figure 1 is a diagram showing the effect of the polyhydroxybutyrate (PHB) synthesis operon on isobutanol production. BLISO and BLpLISO strains were cultured for 72 h for comparative analysis of isobutanol and cell growth. A: Effect of PHB operon on cell growth of transformed Escherichia coli ( E. coli ). B: Effect of PHB operon on isobutanol productivity. Error bars represent the standard deviation of three replicate experiments.
Figure 2 is a schematic diagram of the co-production method of isobutanol and PHB.
Figure 3 is a diagram showing growth profiling of BL21(DE3) and BLpL.
Figure 4 shows the results of confirming the optimal concentration for co-production of isobutanol and PHB in E. coli transformed using various yeast extract concentrations. A: Cell growth of transformed E. coli in the presence of various amounts of yeast extract in culture medium. B: Isobutanol and PHB productivity of transformed E. coli in the presence of various amounts of yeast extract in culture medium. Error bars represent the standard deviation of three replicate experiments.
Figure 5 is a diagram confirming the effect of cofactor engineering on isobutanol and PHB co-production. A: NAD(P)H cost in isobutanol and PHB production pathways. B, C, D: Effect of cofactor engineering on cell growth (B), isobutanol productivity (C) and PHB productivity (D) of transformed E. coli . Error bars represent the standard deviation of three replicate experiments.
Figure 6 is a diagram showing the isobutanol and PHB production capacity of BLISO and BLpLISOpntAB strains during flask culture. BLISO and BLpLISOpntAB strains were cultured for 72 h and analyzed for cell growth (A), isobutanol and PHB production (B), residual glucose detection (C), and acetate content over time. Error bars represent the standard deviation of three replicate experiments.
Figure 7 is a diagram confirming the growth curves of BLISO and BLpLISOpntAB strains under solvent stress. Growth of BLISO and BLpLISOpntAB strains was compared under stress by ethanol (A), isopropanol (B), isobutanol (C), or n-butanol (D). Abbreviations: EtOH, Ethanol; IPA, isopropanol; IBOH, isobutanol; NBA, N-butanol. Error bars represent the standard deviation of three replicate experiments.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Preparation of strains for simultaneous production of isobutanol and polyhydroxybutyrate (PHB)

1-1. Construction of strains containing the isobutanol synthesis operon

All Escherichia coli ( E. coli ) were cultured in LB (lysogeny broth) medium. To construct the plasmid, the heatshock method was used and E. coli DH5α was used as a cloning host. All PCR reactions and ligation were performed using nPfu-Forte DNA polymerase and T4 DNA ligase.

To construct pRSFISO, use a start codon with TTG modified to ATG in Bacillus subtilis ( B. subtilis ) From 168, alsS (acetolactate synthase) (SEQ ID NO: 1) was amplified using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24. Then, pRSFDuet-1 and amplified alsS were digested with BamHI and SacI and ligated. Next, kivD (alpha-ketoisovalerate decarboxylase) (SEQ ID NO: 3) from Lactococcus lactis subsp. lactis KF147, L. lactis subsp. lactis KF147 was prepared using the primer pair of SEQ ID NO: 25 and SEQ ID NO: 26. It was amplified using and further cloned into NdeI and XhoI sites. ilvC (ketol-acid reductoisomerase) (SEQ ID NO: 5) using E. coli MG1655 as a template was amplified using the primer pair of SEQ ID NO: 27 and SEQ ID NO: 28, then introduced using SacI and SalI restriction enzymes, and ilvD ( dihydroxyacid dehydratase (SEQ ID NO: 7) was amplified using the primer pair of SEQ ID NO: 29 and SEQ ID NO: 30 and then introduced using SalI and NotI restriction enzymes. Through this, alsS of B. subtilis 168 and L. lactis subsp were added to pRSFDuet-1 . The vector pRSFISO was constructed into which the isobutanol synthesis pathway was introduced, including kivD from lactis KF147 and ilvCD ( ilvC and ilvD) from E. coli MG1655.

Next, for the generation of the cofactor modulation system, the pACYCDuet-1 plasmid and the primer pair of SEQ ID NO: 31 and SEQ ID NO: 32 and the primer pair of SEQ ID NO: 39 and SEQ ID NO: 40 were used to generate the pntAB (pyridine nucleotide) of E. coli MG1655. transhydrogenase) (SEQ ID NO: 9) and L. lactis subsp. NoxE (NADH oxidase) (SEQ ID NO: 21) of lactis KF147 was amplified and cloned into pACYCDuet-1 using BamHI and SacI restriction enzymes, respectively, to construct vectors pHM55 and pHS12. In addition, fdh (formate dehydrogenase) (SEQ ID NO: 17) of Candida boidinii ( Candida boidinii , C. boidinii ) was amplified using the primer pair of SEQ ID NO: 35 and SEQ ID NO: 36, and then introduced into pACYCDuet-1 at NdeI and XhoI sites. Vector pHS13 was created.

Next, using the primer pair of SEQ ID NO: 35 and SEQ ID NO: 36 and the primer pair of SEQ ID NO: 37 and SEQ ID NO: 38, fdh or yfjB (NAD kinase) (SEQ ID NO: 19) of E. coli MG1655 was amplified, respectively, and then amplified at pHM55. Combination vectors pHS14 and pHS16 were constructed by introducing them into the NdeI and XhoI sites, respectively.

The vector constructed above was transformed into E. coli BL21(DE3) to produce transformants for isobutanol and PHB production. Each transformant was incubated with 0.1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) in M9 minimal medium containing 5% (w/v%) glucose and 0.3% (w/v%) yeast extract at an initial pH of 6.8. was cultured. Aliquots were removed from the culture intermittently and harvested. Glucose and organic acids were quantified by high-pressure liquid chromatography with UV-vis and refractive index detectors. Gas chromatographic analysis (GC-FID) using a flame ionization detector (FID) was performed to quantify isobutanol.

1-2. Introduction of the PHB synthesis operon into a strain containing the isobutanol synthesis operon.

bktB (beta-ketothiolase) (SEQ ID NO: 11) and phaB (acetoacetyl-CoA reductase) under the trc promoter of Ralstonia eutropha ( R. eutropha ) H16 in the transformant prepared in Example 1-1. (SEQ ID NO: 13) and phaC (poly(3-hydroxybutyrate) polymerase) (SEQ ID NO: 15) with the primer pair of SEQ ID NO: 33 and SEQ ID NO: 34, the primer pair of SEQ ID NO: 41 and SEQ ID NO: 42, and SEQ ID NO: 43 and It was amplified using the primer pair of SEQ ID NO: 44, and vector pLW487 containing the isobutanol synthesis pathway genes including each of the amplified genes was introduced to create a strain containing both the PHB synthesis operon and the isobutanol synthesis operon (vectors pLW487 and BLpL strain containing pRSFISO, BLpLISOACYC strain containing vectors pLW487, pRSFISO and pACYCDuet-1, BLpLISOn strain containing vectors pLW487, pRSFISO and pHS12, BLpLISOfdh strain containing vectors pLW487, pRSFISO and pHS13, vectors pLW487, pRSFISO and BLpLISOpp strain containing pHS14, BLpLISOyp strain containing vectors pLW487, pRSFISO and pHS16, BLpLISOpntAB strain containing vectors pLW487, pRSFISO and pHM55) were constructed, The effect of the PHB synthetic operon on isobutanol production was confirmed.

As a result of culturing each of the above strains, cell growth was activated by the additional PHB synthesis operon of R. eutropha H16 (Figure 1A), and isobutanol production was increased approximately 1.6-fold (1.14 g/L) in the engineered E. coli (Figure 1A). 1B).

PHB and isobutanol were found in different locations (intracellular and extracellular, respectively) during the fermentation process (Figure 2). Due to this property, it was confirmed that no additional process to separate the product was required when fermentation was completed.

Additionally, the isobutanol productivity conferred to E. coli by the PHB synthesis operon is generally proportional to cell growth, and the PHB synthesis operon was shown to activate cell activity (Figure 3).

The above results show that expression of the PHB synthetic operon improved isobutanol production.

The plasmids and strains disclosed herein are shown in Table 1 below.

Example 2. Analysis of optimal yeast extract concentration for co-production of PHB and isobutanol

PHB synthesis is critically influenced by the nitrogen source in the medium, and especially when cells enter the nitrogen-limited growth phase, the PHB synthesis operon is activated and polymer production begins. On the other hand, nitrogen-rich compounds such as yeast extract are required for optimal isobutanol production. In other words, since both PHB and isobutanol are sensitive to nitrogen sources, the concentration of yeast extract required for production was optimized.

As a result, because yeast extract is rich in nutrients, cell growth and isobutanol titer were proportional to the yeast extract concentration added to the medium (Figure 4A and B). In 0.3%v/v yeast extract, the previous method (Bhatia, S.K., et al. 2019. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) production from engineered Ralstonia eutropha using synthetic and anaerobically digested food waste derived volatile fatty acids. Int 1.4 and 0.2 g/L of isobutanol and PHB, respectively, were produced as confirmed by GC-FID using J. Biol. Macromol. 133, 1-10.), but these levels were achieved at a yeast extract concentration of 0.3%v When it was higher than /v, it gradually decreased (Figure 4B). Therefore, the yeast extract concentration for co-production of isobutanol and PHB was used as 0.3%v/v in the subsequent examples.

Example 3. NADP(H) manipulation by cofactor engineering for co-production of PHB and isobutanol

The metabolic pathways of isobutanol and PHB synthesis required 2 mol and 1 mol of NAD(P)H, respectively (Figure 5A). Specifically, IlvC and ADH in the isobutanol pathway and PhaB in the PHB synthesis pathway are known to utilize NAD(P)H as a cofactor for each reaction. Therefore, overexpression of both isobutanol and PHB synthetic operons may lead to redox imbalance, which in turn may limit productivity.

Among many potential candidates, fdh from C. boidinii , pntAB and yfjB from E. coli , L. lactis subsp. HO-forming noxE from lactis KF147 was overexpressed as a driving force for cofactor formation.

Applying single or combined expression of the cofactor regeneration systems to examine cell growth and isobutanol and PHB production resulted in increased cell growth for all strains, but was especially high for the BLpLISOpntAB and BLpLISONoxE strains (Figure 5B).

In the case of fdh , it was analyzed to have a detrimental effect on isobutanol and PHB production because the expression resulted in a decrease in isobutanol and PHB synthesis (Figure 5C, D). In addition, isobutanol production increased in all strains except those expressing fdh and noxE (Figure 4C).

In the case of noxE , the gene product is known to activate glucose utilization by manipulating the NADH to NAD+ ratio to compensate for the limited function of the electron transport chain, which can increase PHB production by directing metabolic flux toward acetyl-CoA production. there is. Therefore, noxE seemed to increase the robustness of cell growth but did not increase isobutanol titer, which was analyzed to be because noxE produces NAD+ as its main product. However, this form of cofactor was not a desirable source for isobutanol production and only increased cell growth and PHB production.

In conclusion, pntAB was found to play an active role in promoting cell growth and isobutanol (2.7 g/L) and PHB synthesis (0.52 g/L), confirming that it is suitable for co-production of isobutanol and PHB.

Example 5. Isobutanol production due to increased cell activity by PHB and cofactor engineering

Co-production of isobutanol and PHB was performed to monitor cell growth, isobutanol and PHB production, glucose consumption and acetate accumulation.

During 72 hours of culture, the BLpLISOpntAB strain showed faster cell growth and higher yield (Figure 6A). Isobutanol and PHB production were approximately 2.4 g/L and 0.25 g/L, respectively, which were two times higher than that of the BLISO strain containing only the vector pRSFISO (Figure 6B). Moreover, glucose consumption was slightly faster and more complete, and acetate accumulation was maintained below 0.5 g/L in cultures of the BLpLISOpntAB strain (Figure 6C,D).

These results indicated that the isobutanol and PHB co-producing strains were robust. The PHB synthesis pathway uses acetyl-CoA for PHB production, which prevented acetate overflow when glucose was the only carbon source.

Moreover, maintaining low acetate concentrations in the culture medium increased the utilization of glucose as evidenced by glucose consumption. As a result, it was confirmed that the high metabolic activity and cell growth of E. coli are advantageous for the co-production of isobutanol and PHB.

Example 6. Increased isobutanol tolerance assay by PHB synthesis pathway expression

Solvent toxicity tests were performed including ethanol (40 g/L), isopropanol (20 g/L), isobutanol (10 g/L), and n-butanol (10 g/L), which can promote osmotic shock of the strain.

As a result, although the growth rate was slowed in the early stages due to the metabolic burden caused by the introduced gene, the growth of the PHB-producing strain maintained a significantly high level as the culture time progressed even under solvent shock conditions (Figure 7A -D). However, in the presence of n-butanol, cell growth was severely restricted or stopped for 5 days compared to that for the BLISO strain.

Next, at a specific time point (100 hours), we analyzed whether the PHB synthesis operon activated cell growth or biomass production by determining the number of colony forming units, and found that, in fact, the number of viable cells was not increased by the PHB synthesis operon. (Table 2), an increase in optical density occurred due to an increase in biomass due to PHB accumulation.

Overall, cell growth was increased by the PHB modular system in relation to the amount of biomass at the end of fermentation, thus confirming that PHB synthesis can increase the tolerance of engineered E. coli to isobutanol stress.

The above results confirmed that the isobutanol and PHB co-producing strain according to the present invention has increased cell activity, faster glucose utilization, lower acetate accumulation in the culture medium, and does not require additional processing to separate the final product. .

From the results of the above examples, the isobutanol and PHB co-producing strain according to the present invention is economical by simultaneously utilizing culture medium and cell mass, exhibits increased cell activity and faster glucose utilization, improves strain robustness, and has It can be applied to the industrial production of isobutanol and PHB, as it can reduce acetate accumulation during fermentation without deletion and does not require additional processes to separate the final product.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

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catctgcggt cgatttcgga 1500 aatatcgata t cgtgaaata tgcggaaagc ttcggagcaa ctggcttgcg cgtagaatca 1560 ccagaccagc tggcagatgt tctgcgtcaa ggcatgaacg ctgaaggtcc tgtcatcatc 1620 gatgtcccgg ttgactacag tgataacatt aatttagcaa gtgacaagct tccgaaagaa 1680 ttcggggaac tcatgaaaac gaaagctctc tag 1713 <210> 2 <211> 570 <212> PRT <213> Unknown <220> <223> alsS <40 0> 2 Met Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg Gly 1 5 10 15 Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His Val 20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu Gln 35 40 45 Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala Ala 50 55 60 Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val Val 65 70 75 80 Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu Leu 85 90 95 Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn Val 100 105 110 Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn Ala 115 120 125 Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp Val 130 135 140 Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160 Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val Asn 165 170 175 Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys Leu 180 185 190 Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile Gln 195 200 205 Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg Pro 210 215 220 Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu Pro 225 230 235 240 Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu Glu 245 250 255 Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly Asp 260 265 270 Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp Pro 275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr Ile 290 295 300 Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln Pro 305 310 315 320 Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile Glu 325 330 335 His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Leu 340 345 350 Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala Asp 355 360 365 Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu Arg 370 375 380 Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr Leu 405 410 415 Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala 420 425 430 Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val Ser 435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val 450 455 460 Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser Ala 485 490 495 Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly 500 505 510 Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val Leu 515 520 525 Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val 530 535 540 Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys Glu 545 550 555 560 Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 <210> 3 <211> 1647 <212> DNA <213> Unknown <220> <223> kivD < 18 0 cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240 aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300 acatcaaaag tccaaaatga aggaaaaattt gttcatcata cgctggctga cggtgatttt 360 aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420 aatgcaaccg ttgaaattga ccgagtactt tctgcactac taaaagaaag aaaacctgtc 480 tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540 aaaaaagaaa atccaacttc aaatacaagt gaccaagaga ttttgaataa a attcaagaa 600 agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagctttggc 660 ttagaaaata cagtcactca atttatttca aagacaaaac tccctattac gacattaaac 720 tttggaaaaaa gttcagttga tgaaactctc ccttcatttt taggaatcta taatggtaaa 780 ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840 gttaaactca cagactcttc aacaggagca tttacccatc atttaaatga aaataaaatg 900 atttcactga acatagacga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960 gaatccctca tctcctctct cttagaccta agcggaatag aatacaaagg aaaatatatc 1020 gataaaa agc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080 caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140 ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttatgg tcaaccctta 1 200 tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260 agcagacacc ttttatttat tggtgatggt tcacttcaac ttacagtgca agaattagga 1320 ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380 gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440 tcaaaatta c cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500 actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560 tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggtaaa 1620 ctatttgctg aacaaaaataa atcataa 1647 <210> 4 <211> 548 <212> PRT <213> Unknown <220> <223> kivD <400> 4 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Pro Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Asn Thr 210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Thr Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Ser Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Gly Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Glu Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Ala Lys 515 520 525 Glu Asp Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545 <210> 5 <211> 1476 <212> DNA <213> Unknown <220> <223> ilvC <400> 5 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg a accagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgatt gccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgca g gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaac gatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgatt ccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 138 0 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210> 6 <211> 491 <212> PRT <213> Unknown <220> <223> ilvC <400> 6 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln 1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala 20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser 50 55 60 Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg 65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Glu Leu Ile 85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser 100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu 115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg 130 135 140 Lys Asp Ile Thr Val Val Met Val Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys 180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser 195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys Gln Gly Gly Ile Thr Leu 260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu 275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met 290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln 340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu 420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile 435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala 450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly 485 490 <210> 7 <211> 1851 <212> DNA <213> Unknown <220> <223> ilvD <400> 7 atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60 ctgtggcgcg c caccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120 aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180 gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240 gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cga actgatc 300 gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360 aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420 atctttgttt ccggcggccc gatggaggcc gggaaaacca aact ttccga tcagatcatc 480 aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540 agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600 gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660 ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcat tgtt 720 gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780 agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840 accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900 atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960 taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020 cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080 ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 11 40 ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200 gacgacgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260 ggcctggcgg tgctctacgg taactttgcg gaaaacggct g catcgtgaa aacggcaggc 1320 gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380 gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440 gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500 tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560 tctggt cttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620 attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680 agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaag cctgg 1740 acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800 accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 1851 <210> 8 <211> 616 <212> PRT <213> Unknown <220> <223> ilvD <400> 8 Met Pro Lys Tyr Arg Ser Ala Thr Thr Thr His Gly Arg Asn Met Ala 1 5 10 15 Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe 20 25 30 Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro 35 40 45 Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile 50 55 60 Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser 85 90 95 Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys 100 105 110 Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125 Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser 130 135 140 Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile 145 150 155 160 Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val 165 170 175 Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys 180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr 195 200 205 Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr 210 215 220 His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val 225 230 235 240 Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro 245 250 255 Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp 260 265 270 Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala 275 280 285 Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu 290 295 300 Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys 305 310 315 320 Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu 325 330 335 Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val 340 345 350 Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu 355 360 365 Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly 370 375 380 Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu 385 390 395 400 Asp Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr 405 410 415 Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn 420 425 430 Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe 435 440 445 Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala 450 455 460 Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Val Ile Arg Tyr 465 470 475 480 Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr 485 490 495 Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr 500 505 510 Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val 515 520 525 Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly 530 535 540 Asp Leu Ile Ala Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val 545 550 555 560 Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly 565 570 575 Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala 580 585 590 Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val 595 600 605 Arg Asp Lys Ser Lys Leu Gly Gly 610 615 <210> 9 <211> 2932 <212> DNA <213> Unknown <220> <223> pntAB <400> 9 atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60 aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120 caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180 aatagcgtct ggca gtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240 gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300 ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 36 0 atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420 gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480 gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540 attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600 aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660 ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720 ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780 aaaccag cgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840 attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900 ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960 acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020 gagaaagacg gcaatatcac tgttgatttt gatgatg tgg tgattcgcgg cgtgaccgtg 1080 atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140 gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200 cgtaaatacg cgttgatgg c gctggcaatc attctttttg gctggatggc aagcgttgcg 1260 ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320 gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380 tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440 cttagtttta tcgcggtgct tatagccagc attaatatt t tcggtggctt caccgtgact 1500 cagcgcatgc tgaaaatgtt ccgcaaaaat taaggggtaa catatgtctg gaggattagt 1560 tacagctgca tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa 1620 acatgaaacg tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat 1680 cgcaaccatt tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat 1740 tggtggggca attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact 1800 ggtggcgatc ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta 1860 tctgcatcat gacgcgggaa tggcaccgat tctggtcaat attcacctga cgg aagtgtt 1920 cctcggtatc ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact 1980 gtgtggcaag atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc 2040 ggctctggtc gtttccttcc tgctgct gat tgtatttgtt cgcacggaca gcgtcggcct 2100 gcaagtgctg gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt 2160 cgcctccatc ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg 2220 ctgggcggct gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc 2280 gctggtcggt tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaac cgttc 2340 ctttatcagc gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca 2400 ggaagtgggt gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc 2460 ccattcagtg atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt 2520 cgctgaaatt actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc 2580 ggtcgcgggg cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta 2640 tgacatcgtg ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact 2700 ggtgattggt gctaacgata cggttaaccc ggcggcgcag gatgatccga agagtccgat 2760 tgctgg tatg cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc 2820 gatgaacact ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat 2880 gctgtttggt gacgccaaag ccagcgtgga tgcaatcctg aaagctctg t aa 2932 <210> 10 <211> 973 <212 > PRT <213> Unknown <220> <223> pntAB <400> 10 Met Arg Ile Gly Ile Pro Arg Glu Arg Leu Thr Asn Glu Thr Arg Val 1 5 10 15 Ala Ala Thr Pro Lys Thr Val Glu Gln Leu Leu Lys Leu Gly Phe Thr 20 25 30 Val Ala Val Glu Ser Gly Ala Gly Gln Leu Ala Ser Phe Asp Asp Lys 35 40 45 Ala Phe Val Gln Ala Gly Ala Glu Ile Val Glu Gly Asn Ser Val Trp 50 55 60 Gln Ser Glu Ile Ile Leu Lys Val Asn Ala Pro Leu Asp Asp Glu Ile 65 70 75 80 Ala Leu Leu Asn Pro Gly Thr Thr Leu Val Ser Phe Ile Trp Pro Ala 85 90 95 Gln Asn Pro Glu Leu Met Gln Lys Leu Ala Glu Arg Asn Val Thr Val 100 105 110 Met Ala Met Asp Ser Val Pro Arg Ile Ser Arg Ala Gln Ser Leu Asp 115 120 125 Ala Leu Ser Ser Met Ala Asn Ile Ala Gly Tyr Arg Ala Ile Val Glu 130 135 140 Ala Ala His Glu Phe Gly Arg Phe Phe Thr Gly Gln Ile Thr Ala Ala 145 150 155 160 Gly Lys Val Pro Pro Ala Lys Val Met Val Ile Gly Ala Gly Val Ala 165 170 175 Gly Leu Ala Ala Ile Gly Ala Ala Asn Ser Leu Gly Ala Ile Val Arg 180 185 190 Ala Phe Asp Thr Arg Pro Glu Val Lys Glu Gln Val Gln Ser Met Gly 195 200 205 Ala Glu Phe Leu Glu Leu Asp Phe Lys Glu Glu Ala Gly Ser Gly Asp 210 215 220 Gly Tyr Ala Lys Val Met Ser Asp Ala Phe Ile Lys Ala Glu Met Glu 225 230 235 240 Leu Phe Ala Ala Gln Ala Lys Glu Val Asp Ile Ile Val Thr Thr Ala 245 250 255 Leu Ile Pro Gly Lys Pro Ala Pro Lys Leu Ile Thr Arg Glu Met Val 260 265 270 Asp Ser Met Lys Ala Gly Ser Val Ile Val Asp Leu Ala Ala Gln Asn 275 280 285 Gly Gly Asn Cys Glu Tyr Thr Val Pro Gly Glu Ile Phe Thr Thr Glu 290 295 300 Asn Gly Val Lys Val Ile Gly Tyr Thr Asp Leu Pro Gly Arg Leu Pro 305 310 315 320 Thr Gln Ser Ser Gln Leu Tyr Gly Thr Asn Leu Val Asn Leu Leu Lys 325 330 335 Leu Leu Cys Lys Glu Lys Asp Gly Asn Ile Thr Val Asp Phe Asp Asp 340 345 350 Val Val Ile Arg Gly Val Thr Val Ile Arg Ala Gly Glu Ile Thr Trp 355 360 365 Pro Ala Pro Pro Ile Gln Val Ser Ala Gln Pro Gln Ala Ala Gln Lys 370 375 380 Ala Ala Pro Glu Val Lys Thr Glu Glu Lys Cys Thr Cys Ser Pro Trp 385 390 395 400 Arg Lys Tyr Ala Leu Met Ala Leu Ala Ile Ile Leu Phe Gly Trp Met 405 410 415 Ala Ser Val Ala Pro Lys Glu Phe Leu Gly His Phe Thr Val Phe Ala 420 425 430 Leu Ala Cys Val Val Gly Tyr Tyr Val Val Trp Asn Val Ser His Ala 435 440 445 Leu His Thr Pro Leu Met Ser Val Thr Asn Ala Ile Ser Gly Ile Ile 450 455 460 Val Val Gly Ala Leu Leu Gln Ile Gly Gln Gly Gly Trp Val Ser Phe 465 470 475 480 Leu Ser Phe Ile Ala Val Leu Ile Ala Ser Ile Asn Ile Phe Gly Gly 485 490 495 Phe Thr Val Thr Gln Arg Met Leu Lys Met Phe Arg Lys Asn His Met 500 505 510 Ser Gly Gly Leu Val Thr Ala Ala Tyr Ile Val Ala Ala Ile Leu Phe 515 520 525 Ile Phe Ser Leu Ala Gly Leu Ser Lys His Glu Thr Ser Arg Gln Gly 530 535 540 Asn Asn Phe Gly Ile Ala Gly Met Ala Ile Ala Leu Ile Ala Thr Ile 545 550 555 560 Phe Gly Pro Asp Thr Gly Asn Val Gly Trp Ile Leu Leu Ala Met Val 565 570 575 Ile Gly Gly Ala Ile Gly Ile Arg Leu Ala Lys Lys Val Glu Met Thr 580 585 590 Glu Met Pro Glu Leu Val Ala Ile Leu His Ser Phe Val Gly Leu Ala 595 600 605 Ala Val Leu Val Gly Phe Asn Ser Tyr Leu His His Asp Ala Gly Met 610 615 620 Ala Pro Ile Leu Val Asn Ile His Leu Thr Glu Val Phe Leu Gly Ile 625 630 635 640 Phe Ile Gly Ala Val Thr Phe Thr Gly Ser Val Val Ala Phe Gly Lys 645 650 655 Leu Cys Gly Lys Ile Ser Ser Lys Pro Leu Met Leu Pro Asn Arg His 660 665 670 Lys Met Asn Leu Ala Ala Leu Val Val Ser Phe Leu Leu Leu Ile Val 675 680 685 Phe Val Arg Thr Asp Ser Val Gly Leu Gln Val Leu Ala Leu Leu Ile 690 695 700 Met Thr Ala Ile Ala Leu Val Phe Gly Trp His Leu Val Ala Ser Ile 705 710 715 720 Gly Gly Ala Asp Met Pro Val Val Val Ser Met Leu Asn Ser Tyr Ser 725 730 735 Gly Trp Ala Ala Ala Ala Ala Ala Gly Phe Met Leu Ser Asn Asp Leu Leu 740 745 750 Ile Val Thr Gly Ala Leu Val Gly Ser Ser Gly Ala Ile Leu Ser Tyr 755 760 765 Ile Met Cys Lys Ala Met Asn Arg Ser Phe Ile Ser Val Ile Ala Gly 770 775 780 Gly Phe Gly Thr Asp Gly Ser Ser Thr Gly Asp Asp Gln Glu Val Gly 785 790 795 800 Glu His Arg Glu Ile Thr Ala Glu Glu Thr Ala Glu Leu Leu Lys Asn 805 810 815 Ser His Ser Val Ile Ile Thr Pro Gly Tyr Gly Met Ala Val Ala Gln 820 825 830 Ala Gln Tyr Pro Val Ala Glu Ile Thr Glu Lys Leu Arg Ala Arg Gly 835 840 845 Ile Asn Val Arg Phe Gly Ile His Pro Val Ala Gly Arg Leu Pro Gly 850 855 860 His Met Asn Val Leu Leu Ala Glu Ala Lys Val Pro Tyr Asp Ile Val 865 870 875 880 Leu Glu Met Asp Glu Ile Asn Asp Asp Phe Ala Asp Thr Asp Thr Val 885 890 895 Leu Val Ile Gly Ala Asn Asp Thr Val Asn Pro Ala Ala Gln Asp Asp 900 905 910 Pro Lys Ser Pro Ile Ala Gly Met Pro Val Leu Glu Val Trp Lys Ala 915 920 925 Gln Asn Val Ile Val Phe Lys Arg Ser Met Asn Thr Gly Tyr Ala Gly 930 935 940 Val Gln Asn Pro Leu Phe Phe Lys Glu Asn Thr His Met Leu Phe Gly 945 950 955 960 Asp Ala Lys Ala Ser Val Asp Ala Ile Leu Lys Ala Leu 965 970 <210> 11 <211> 1185 <212> DNA <213> Unknown <220> <223> bktB <400> 11 atgacgcgtg aagtggtagt ggtaagcggt gtccg taccg cgatcgggac ctttggcggc 60 agcctgaagg atgtggcacc ggcggagctg ggcgcactgg tggtgcgcga ggcgctggcg 120 cgcgcgcagg tgtcgggcga cgatgtcggc cacgtggtat tcggcaacgt gatccagacc 180 gagccgcgcg acatg tatct gggccgcgtc gcggccgtca acggcggggt gacgatcaac 240 gcccccgcgc tgaccgtgaa ccgcctgtgc ggctcgggcc tgcaggccat tgtcagcgcc 300 gcgcagacca tcctgctggg cgataccgac gtcgccatcg gcggcggcgc ggaaag catg 360 agccgcgcac cgtacctggc gccggcagcg cgctggggcg cacgcatggg cgacgccggc 420 ctggtcgaca tgatgctggg tgcgctgcac gatcccttcc atcgcatcca catgggcgtg 480 accgccgaga atgtcgccaa ggaatacgac atctcgcgcg cgcagcagga cgaggccgcg 540 ctggaatcgc accgccgcgc ttcggcagcg atcaaggccg gctacttcaa ggaccagatc 600 gtcccggtgg tgagcaaggg ccgcaagggc gacgtgacct tcgacaccga cgagcacgtg 660 cgccatgacg ccaccatcga cgacatgacc aagctcaggc cggtcttcgt caaggaaaac 720 ggcacggtca cggccggcaa tgcctcgggc ctgaacgacg ccgccgccgc ggtggtgatg 780 atggagcgcg cc gaagccga gcgccgcggc ctgaagccgc tggcccgcct ggtgtcgtac 840 ggccatgccg gcgtggaccc gaaggccatg ggcatcggcc cggtgccggc gacgaagatc 900 gcgctggagc gcgccggcct gcaggtgtcg gacctggacg tgatcgaagc caacgaagcc 960 tttgccgcac aggcgtgcgc cgtgaccaag gcgctcggtc tggacccggc caaggttaac 1020 ccgaacggct cgggcatctc gctgggccac ccgatcggcg c caccggtgc cctgatcacg 1080 gtgaaggcgc tgcatgagct gaaccgcgtg cagggccgct acgcgctggt gacgatgtgc 1140 atcggcggcg ggcagggcat tgccgccatc ttcgagcgta tctga 1185 <210> 12 <211> 394 <2 12> PRT <213> Unknown <220> <223> bktB <400> 12 Met Thr Arg Glu Val Val Val Val Ser Gly Val Arg Thr Ala Ile Gly 1 5 10 15 Thr Phe Gly Gly Ser Leu Lys Asp Val Ala Pro Ala Glu Leu Gly Ala 20 25 30 Leu Val Val Arg Glu Ala Leu Ala Arg Ala Gln Val Ser Gly Asp Asp 35 40 45 Val Gly His Val Val Phe Gly Asn Val Ile Gln Thr Glu Pro Arg Asp 50 55 60 Met Tyr Leu Gly Arg Val Ala Ala Val Asn Gly Gly Val Thr Ile Asn 65 70 75 80 Ala Pro Ala Leu Thr Val Asn Arg Leu Cys Gly Ser Gly Leu Gln Ala 85 90 95 Ile Val Ser Ala Ala Gln Thr Ile Leu Leu Gly Asp Thr Asp Val Ala 100 105 110 Ile Gly Gly Gly Ala Glu Ser Met Ser Arg Ala Pro Tyr Leu Ala Pro 115 120 125 Ala Ala Arg Trp Gly Ala Arg Met Gly Asp Ala Gly Leu Val Asp Met 130 135 140 Met Leu Gly Ala Leu His Asp Pro Phe His Arg Ile His Met Gly Val 145 150 155 160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Asp Ile Ser Arg Ala Gln Gln 165 170 175 Asp Glu Ala Ala Leu Glu Ser His Arg Arg Ala Ser Ala Ala Ile Lys 180 185 190 Ala Gly Tyr Phe Lys Asp Gln Ile Val Pro Val Val Ser Lys Gly Arg 195 200 205 Lys Gly Asp Val Thr Phe Asp Thr Asp Glu His Val Arg His Asp Ala 210 215 220 Thr Ile Asp Asp Met Thr Lys Leu Arg Pro Val Phe Val Lys Glu Asn 225 230 235 240 Gly Thr Val Thr Ala Gly Asn Ala Ser Gly Leu Asn Asp Ala Ala Ala 245 250 255 Ala Val Val Met Met Glu Arg Ala Glu Ala Glu Arg Arg Gly Leu Lys 260 265 270 Pro Leu Ala Arg Leu Val Ser Tyr Gly His Ala Gly Val Asp Pro Lys 275 280 285 Ala Met Gly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu Glu Arg 290 295 300 Ala Gly Leu Gln Val Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala 305 310 315 320 Phe Ala Ala Gln Ala Cys Ala Val Thr Lys Ala Leu Gly Leu Asp Pro 325 330 335 Ala Lys Val Asn Pro Asn Gly Ser Gly Ile Ser Leu Gly His Pro Ile 340 345 350 Gly Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu Leu Asn 355 360 365 Arg Val Gln Gly Arg Tyr Ala Leu Val Thr Met Cys Ile Gly Gly Gly 370 375 380 Gln Gly Ile Ala Ala Ile Phe Glu Arg Ile 385 390 <210> 13 <211> 741 <212> DNA <213> Unknown <220> <223> phaB <400> 13 atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60 cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120 cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180 aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240 gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 3 00 acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360 aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420 gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480 catggcttca ccatggcact ggcgca ggaa gtggcgacca agggcgtgac cgtcaacacg 540 gtctctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600 aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660 tgcg cctggt tgtcgtcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720 ggcggcctgc atatgggctg a 741 <210> 14 <211> 246 <212> PRT <213> Unknown <220> <223> phaB <400> 14 Met Thr Gln Arg Ile Ala Tyr Val Thr Gly Gly Met Gly Gly Ile Gly 1 5 10 15 Thr Ala Ile Cys Gln Arg Leu Ala Lys Asp Gly Phe Arg Val Val Ala 20 25 30 Gly Cys Gly Pro Asn Ser Pro Arg Arg Glu Lys Trp Leu Glu Gln Gln 35 40 45 Lys Ala Leu Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn Val Ala Asp 50 55 60 Trp Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser Glu Val Gly 65 70 75 80 Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val 85 90 95 Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn 100 105 110 Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala 115 120 125 Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn Gly Gln 130 135 140 Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala Lys Ala Gly Leu 145 150 155 160 His Gly Phe Thr Met Ala Leu Ala Gln Glu Val Ala Thr Lys Gly Val 165 170 175 Thr Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp Met Val Lys 180 185 190 Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr Ile Pro Val 195 200 205 Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu 210 215 220 Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn 225 230 235 240 Gly Gly Leu His Met Gly 245 <210> 15 <211> 1770 <212> DNA <213> Unknown <220> <223> phaC <400> 15 atggcgaccg gcaaaggcgc ggcagcttcc acgcaggaag gcaagtccca accattcaag 60 gtcacgccgg ggccattcga tccagccaca tggctggaat ggtcccgcca gtggcagggc 120 actgaaggca acggccacgc ggccgcgtcc ggcattccgg gcctggatgc gctggca ggc 180 gtcaagatcg cgccggcgca gctgggtgat atccagcagc gctacatgaa ggacttctca 240 gcgctgtggc aggccatggc cgagggcaag gccgaggcca ccggtccgct gcacgaccgg 300 cgcttcgccg gcgacgcatg gcgcaccaac ctcccatatc gcttc gctgc cgcgttctac 360 ctgctcaatg cgcgcgcctt gaccgagctg gccgatgccg tcgaggccga tgccaagacc 420 cgccagcgca tccgcttcgc gatctcgcaa tgggtcgatg cgatgtcgcc cgccaacttc 480 cttgccacca atcccgaggc gcagcgcctg ctgatcgagt cgggcggcga atcgctgcgt 540 gccggcgtgc gcaacatgat ggaagacctg acacgcggca agatctcg ca gaccgacgag 600 agcgcgtttg aggtcggccg caatgtcgcg gtgaccgaag gcgccgtggt cttcgagaac 660 gagtacttcc agctgttgca gtacaagccg ctgaccgaca aggtgcacgc gcgcccgctg 720 ctgatggtgc cgccgtgcat ca acaagtac tacatcctgg acctgcagcc ggagagctcg 780 ctggtgcgcc atgtggtgga gcagggacat acggtgtttc tggtgtcgtg gcgcaatccg 840 gacgccagca tggccggcag cacctgggac gactacatcg agcacgcggc catccgcgcc 900 atcgaagtcg cgcgcgacat cagcggccag gacaagatca acgtgctcgg cttctgcgtg 960 ggcggcacca ttgtctcgac cgcgctggcg gtgctggccg cgcgcggcga gcacccggcc 1 020 gccagcgtca cgctgctgac cacgctgctg gactttgccg acacgggcat cctcgacgtc 1080 tttgtcgacg agggccatgt gcagttgcgc gaggccacgc tgggcggcgg cgccggcgcg 1140 ccgtgcgcgc tgctgcgcgg ccttga gctg gccaatacct tctcgttctt gcgcccgaac 1200 gacctggtgt ggaactacgt ggtcgacaac tacctgaagg gcaacacgcc ggtgccgttc 1260 gacctgctgt tctggaacgg cgacgccacc aacctgccgg ggccgtggta ctgctggtac 1320 ctgcgccaca cctacctgca gaacgagctc aaggtaccgg gcaagctgac cgtgtgcggc 1380 gtgccggtgg acctggccag catcgacgtg ccgacctata tctacggctc gcgcgaagac 1440 catatcgtg c cgtggaccgc ggcctatgcc tcgaccgcgc tgctggcgaa caagctgcgc 1500 ttcgtgctgg gtgcgtcggg ccatatcgcc ggtgtgatca acccgccggc caagaacaag 1560 cgcagccact ggactaacga tgcgctgccg gagtcgccgc agcaatgg ct ggccggcgcc 1620 atcgagcatc acggcagctg gtggccggac tggaccgcat ggctggccgg gcaggccggc 1680 gcgaaacgcg ccgcgcccgc caactatggc aatgcgcgct atcgcgcaat cgaacccgcg 1740 cctgggcgat acgtcaaagc caaggcatga 1770 <210> 16 <211> 589 <212> PRT <213> Unknown <220> <223> phaC <400> 16 Met Ala Thr Gly Lys Gly Ala Ala Ser Thr Gln Glu Gly Lys Ser 1 5 10 15 Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro Ala Thr Trp Leu 20 25 30 Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly His Ala Ala 35 40 45 Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val Lys Ile Ala 50 55 60 Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys Asp Phe Ser 65 70 75 80 Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala Glu Ala Thr Gly Pro 85 90 95 Leu His Asp Arg Arg Phe Ala Gly Asp Ala Trp Arg Thr Asn Leu Pro 100 105 110 Tyr Arg Phe Ala Ala Ala Phe Tyr Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125 Glu Leu Ala Asp Ala Val Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140 Arg Phe Ala Ile Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe 145 150 155 160 Leu Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly 165 170 175 Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg 180 185 190 Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val Gly Arg Asn 195 200 205 Val Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn Glu Tyr Phe Gln 210 215 220 Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val His Ala Arg Pro Leu 225 230 235 240 Leu Met Val Pro Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255 Pro Glu Ser Ser Leu Val Arg His Val Val Glu Gln Gly His Thr Val 260 265 270 Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr 275 280 285 Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile Glu Val Ala 290 295 300 Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val 305 310 315 320 Gly Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly 325 330 335 Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe 340 345 350 Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly His Val Gln 355 360 365 Leu Arg Glu Ala Thr Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu 370 375 380 Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn 385 390 395 400 Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn Thr 405 410 415 Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu 420 425 430 Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn 435 440 445 Glu Leu Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp 450 455 460 Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu Asp 465 470 475 480 His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala 485 490 495 Asn Lys Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val 500 505 510 Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala 515 520 525 Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu His His 530 535 540 Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly 545 550 555 560 Ala Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala 565 570 575 Ile Glu Pro Ala Pro Gly Arg Tyr Val Lys Ala Lys Ala 580 585 <210> 17 <211> 1095 < 212> DNA <213> Unknown <220> <223> fdh <400> 17 atgaaaattg ttctggtgct gtatgatgcc ggcaaacatg cggccgatga agaaaaactg 60 tacggctgca ccgaaaacaa actgggtatc gcaaattggc tgaaagatca gggtcacgaa 120 ctgattacca cgag tgataa agaaggcggt aacagcgtgc tggatcagca tatcccggat 180 gccgatatta tcattaccac gccgtttcac ccggcatata tcacgaaaga acgcattgat 240 aaagcgaaaa aactgaaact ggtggttgtg gcgggcgttg gttctgatca tatcgatctg 300 gattacatta accagaccgg caagaaaatt agcgttctgg aagtgacggg tagcaatgtt 360 gtgtctgtgg cagaacacgt tgtgatgacc atgctggttc tggtgcgtaa ctttgttccg 420 gcgcatgaac agatcattaa tcacgatt gg gaagtggcag cgatcgcgaa agatgcctat 480 gatattgaag gcaaaaccat cgcgacgatt ggcgccggtc gtatcggtta ccgcgttctg 540 gaacgtctgg tgccgttcaa cccgaaagaa ctgctgtatt acgattatca ggccctgccg 600 aaagatgcag aagaaaaagt tggcgcgcgt cgcgtggaaa atatcgaaga actggttgcc 660 caggcagata ttgttaccgt gaacgcgccg ctgcacgcgg gcacgaaagg tctgatcaac 720 aaagaactgc tgagcaaatt caaaaaaggc gcgtggctgg ttaataccgc acgcggtgcg 780 atttgtgttg ccgaagatgt ggccgcagcg ctggaaagcg gtcagctgcg tggttatggc 840 ggtgatgtgt ggttcccgca gcc ggcaccg aaagatcatc cgtggcgtga tatgcgcaac 900 aaatatggcg ccggtaatgc aatgaccccg cactacagcg gcaccacgct ggatgcacag 960 acccgctatg cccagggcac gaaaaacatt ctggaatctt tctttaccgg taaattcgat 1020 taccgtccgc agg atatcat tctgctgaat ggcgaatatg tgacgaaagc gtacggtaaa 1080 cacgataaaa aatta 1095 <210> 18 <211> 365 <212> PRT <213> Unknown <220> <223> fdh <400> 18 Met Lys Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp 1 5 10 15 Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn 20 25 30 Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu 35 40 45 Gly Gly Asn Ser Val Leu Asp Gln His Ile Pro Asp Ala Asp Ile Ile 50 55 60 Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Ile Asp 65 70 75 80 Lys Ala Lys Lys Leu Lys Leu Val Val Val Ala Gly Val Gly Ser Asp 85 90 95 His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile Ser Val 100 105 110 Leu Glu Val Thr Gly Ser Asn Val Val Ser Val Ala Glu His Val Val 115 120 125 Met Thr Met Leu Val Leu Val Arg Asn Phe Val Pro Ala His Glu Gln 130 135 140 Ile Ile Asn His Asp Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr 145 150 155 160 Asp Ile Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly 165 170 175 Tyr Arg Val Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Glu Leu Leu 180 185 190 Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Asp Ala Glu Glu Lys Val Gly 195 200 205 Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile 210 215 220 Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu Ile Asn 225 230 235 240 Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Val Asn Thr 245 250 255 Ala Arg Gly Ala Ile Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu 260 265 270 Ser Gly Gln Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro 275 280 285 Ala Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala 290 295 300 Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln 305 310 315 320 Thr Arg Tyr Ala Gln Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr 325 330 335 Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn Gly Glu 340 345 350 Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys Leu 355 360 365 <210> 19 <211> 879 <212> DNA <213> Unknown <220> <223> yfjB(nadK) <400> 19 atgaataatc atttcaagtg tattggcatt gtgggacacc cacggcaccc cactgcactg 60 acaacacatg aaatgctcta ccgctggctg tgcacaaaag gttacgaggt catcgttgag 120 caacaaatcg ctcacgaact gcaactgaag aatgtgaaaa ctggcacgct cgcggagatt 180 gggcaactag ctgatctcgc ggtagtcgtt ggtggcgacg gtaatatgct gggcgcggca 240 cgcacactcg cccgttacga tattaaagtt attggaatca accgtggcaa cctgggtttc 300 ctgactgacc ttgaccccga taacgcccag caacagttag ccgatgtgct ggaaggccac 360 tacatcagcg agaaacgttt tttgctggaa gcgcaagtct gtcagcaaga ttgccagaaa 420 cgcatcagca ccgcgata aa tgaagtggtg cttcatccag gcaaagtggc gcatatgatt 480 gagttcgaag tgtatatcga cgagatcttt gcgttttctc agcgatctga tggactaatt 540 atttcgacgc caacaggctc caccgcctat tccctctctg caggcggtcc tattctgacc 600 ccctctctgg at gcgattac cctggtgccc atgttcccgc atacgttgtc agcacgacca 660 ctggtcataa acagcagcag cacgatccgt ctgcgttttt cgcatcgccg taacgacctg 720 gaaatcagtt gcgacagcca gatagcactg ccgattcagg aaggtgaaga tgtcctgatt 780 cgtcgctgtg attaccatct gaatctgatt catccgaaag attacagtta tttcaacaca 840 ttaagcacca agctcggctg gtcaaaaaaa tt attctaa 879 <210> 20 <211> 292 <212> PRT <213> Unknown <220> <223> yfjB(nadK) <400 > 20 Met Asn Asn His Phe Lys Cys Ile Gly Ile Val Gly His Pro Arg His 1 5 10 15 Pro Thr Ala Leu Thr Thr His Glu Met Leu Tyr Arg Trp Leu Cys Thr 20 25 30 Lys Gly Tyr Glu Val Ile Val Glu Gln Gln Ile Ala His Glu Leu Gln 35 40 45 Leu Lys Asn Val Lys Thr Gly Thr Leu Ala Glu Ile Gly Gln Leu Ala 50 55 60 Asp Leu Ala Val Val Gly Gly Asp Gly Asn Met Leu Gly Ala Ala 65 70 75 80 Arg Thr Leu Ala Arg Tyr Asp Ile Lys Val Ile Gly Ile Asn Arg Gly 85 90 95 Asn Leu Gly Phe Leu Thr Asp Leu Asp Pro Asp Asn Ala Gln Gln Gln 100 105 110 Leu Ala Asp Val Leu Glu Gly His Tyr Ile Ser Glu Lys Arg Phe Leu 115 120 125 Leu Glu Ala Gln Val Cys Gln Gln Asp Cys Gln Lys Arg Ile Ser Thr 130 135 140 Ala Ile Asn Glu Val Val Leu His Pro Gly Lys Val Ala His Met Ile 145 150 155 160 Glu Phe Glu Val Tyr Ile Asp Glu Ile Phe Ala Phe Ser Gln Arg Ser 165 170 175 Asp Gly Leu Ile Ser Thr Pro Thr Gly Ser Thr Ala Tyr Ser Leu 180 185 190 Ser Ala Gly Gly Pro Ile Leu Thr Pro Ser Leu Asp Ala Ile Thr Leu 195 200 205 Val Pro Met Phe Pro His Thr Leu Ser Ala Arg Pro Leu Val Ile Asn 210 215 220 Ser Ser Ser Thr Ile Arg Leu Arg Phe Ser His Arg Arg Asn Asp Leu 225 230 235 240 Glu Ile Ser Cys Asp Ser Gln Ile Ala Leu Pro Ile Gln Glu Gly Glu 245 250 255 Asp Val Leu Ile Arg Arg Cys Asp Tyr His Leu Asn Leu Ile His Pro 260 265 270 Lys Asp Tyr Ser Tyr Phe Asn Thr Leu Ser Thr Lys Leu Gly Trp Ser 275 280 285 Lys Lys Leu Phe 290 <210> 21 <211> 1341 <212> DNA <213> Unknown <220> <223> noxE <400> 21 atgaaaatcg tagttatcgg tacgaaccac gcaggcattg ctacagcaaa tacatttaatt 60 gatcaatatc caggccatga gattgttatg attgaccgga acagta atat gagttacttg 120 gggtgtggaa cagctatttg ggtcggaaga caaattgaaa aaccagatga gctgttttat 180 gccaaagcag aagatttga aaaaaagggga gtaaagatat taacagaaac agaagtttca 240 gaaattgact ttactaataa aatgatttat gccaagtcaa aaactggaga aaagattaca 300 gaaagttatg ataaactcgt tctggcaaca ggttcacgtc caattattcc taacttgcca 360 ggaaaagatc ttaaaggcat tcatttttta aaactttttc aagaagggca agccattgac 420 gaagagtttg ctaagaatga tgtgaaacgg attgctgtga ttggtgctgg ttatattggg 480 acagaaattg ctgaagctgc caaacgtcgt ggaaaagaag tcctactttt tgat gcagaa 540 agtacttcac ttgcttcata ttatgatgaa gagtttgcta aagggatgga tgaaaatctt 600 gcccaacatg gaattgaact ccattttggg gaattagctc aagagtttaa ggcaaatgaa 660 gaaggtcatg tatcacagat tgtaactaat aaatcaactt atgatgttga cctcgttat 720 aactgtattg gctttacagc caatagtgca ttggctggtg aacatttaga aacctttaaa 78 0 aatggagcaa tcaaagtgaa taaacatcaa caaagtagtg acccagatgt ttatgctgta 840 ggagatgttg ccacaatcta ttctaatgcg ttacaagact tcacctacat tgcccttgcc 900 tcaaacgctg ttcgctcagg gattgttgct ggtcataata ttggaggaaa atcaatagag 960 tctgttggtg tacaaggttc taatggaatc tctatttttg gctacaatat gacttctacg 1020 ggcttgtcgg ttaaagctgc taaaaaaatc ggcctagaag tttcatttag tgattttgaa 1080 gataagcaaa aagcatggtt ccttcatgaa aataatgata gtgtgaaaat tcgtatcgtt 1140 tatgaaacaa aaagtcgcag aattattggt gctcaacttg ctagcaagag tgaaataatt 1200 gca ggaaata ttaatatgtt tagtttagct attcaagaaa agaaaacgat tgatgaatta 1260 gccttacttg atttattttt cttaccacac ttcaatagtc catataatta catgactgtt 1320 gcagctttaa atgcaaaata a 1341 <210> 22 <211> 446 <212> PRT <213> Unknown <220> <223> noxE <400> 22 Met Lys Ile Val Val Ile Gly Thr Asn His Ala Gly Ile Ala Thr Ala 1 5 10 15 Asn Thr Leu Ile Asp Gln Tyr Pro Gly His Glu Ile Val Met Ile Asp 20 25 30 Arg Asn Ser Asn Met Ser Tyr Leu Gly Cys Gly Thr Ala Ile Trp Val 35 40 45 Gly Arg Gln Ile Glu Lys Pro Asp Glu Leu Phe Tyr Ala Lys Ala Glu 50 55 60 Asp Phe Glu Lys Lys Gly Val Lys Ile Leu Thr Glu Thr Glu Val Ser 65 70 75 80 Glu Ile Asp Phe Thr Asn Lys Met Ile Tyr Ala Lys Ser Lys Thr Gly 85 90 95 Glu Lys Ile Thr Glu Ser Tyr Asp Lys Leu Val Leu Ala Thr Gly Ser 100 105 110 Arg Pro Ile Ile Pro Asn Leu Pro Gly Lys Asp Leu Lys Gly Ile His 115 120 125 Phe Leu Lys Leu Phe Gln Glu Gly Gln Ala Ile Asp Glu Glu Phe Ala 130 135 140 Lys Asn Asp Val Lys Arg Ile Ala Val Ile Gly Ala Gly Tyr Ile Gly 145 150 155 160 Thr Glu Ile Ala Glu Ala Ala Lys Arg Arg Gly Lys Glu Val Leu Leu 165 170 175 Phe Asp Ala Glu Ser Thr Ser Leu Ala Ser Tyr Tyr Asp Glu Glu Phe 180 185 190 Ala Lys Gly Met Asp Glu Asn Leu Ala Gln His Gly Ile Glu Leu His 195 200 205 Phe Gly Glu Leu Ala Gln Glu Phe Lys Ala Asn Glu Glu Gly His Val 210 215 220 Ser Gln Ile Val Thr Asn Lys Ser Thr Tyr Asp Val Asp Leu Val Ile 225 230 235 240 Asn Cys Ile Gly Phe Thr Ala Asn Ser Ala Leu Ala Gly Glu His Leu 245 250 255 Glu Thr Phe Lys Asn Gly Ala Ile Lys Val Asn Lys His Gln Gln Ser 260 265 270 Ser Asp Pro Asp Val Tyr Ala Val Gly Asp Val Ala Thr Ile Tyr Ser 275 280 285 Asn Ala Leu Gln Asp Phe Thr Tyr Ile Ala Leu Ala Ser Asn Ala Val 290 295 300 Arg Ser Gly Ile Val Ala Gly His Asn Ile Gly Gly Lys Ser Ile Glu 305 310 315 320 Ser Val Gly Val Gln Gly Ser Asn Gly Ile Ser Ile Phe Gly Tyr Asn 325 330 335 Met Thr Ser Thr Gly Leu Ser Val Lys Ala Ala Lys Lys Ile Gly Leu 340 345 350 Glu Val Ser Phe Ser Asp Phe Glu Asp Lys Gln Lys Ala Trp Phe Leu 355 360 365 His Glu Asn Asn Asp Ser Val Lys Ile Arg Ile Val Tyr Glu Thr Lys 370 375 380 Ser Arg Arg Ile Ile Gly Ala Gln Leu Ala Ser Lys Ser Glu Ile Ile 385 390 395 400 Ala Gly Asn Ile Asn Met Phe Ser Leu Ala Ile Gln Glu Lys Lys Thr 405 410 415 Ile Asp Glu Leu Ala Leu Leu Asp Leu Phe Phe Leu Pro His Phe Asn 420 425 430 Ser Pro Tyr Asn Tyr Met Thr Val Ala Ala Leu Asn Ala Lys 435 440 445 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> alsS F <400> 23 ctctctggat ccgatgacaa aagcaacaaa agaac 35 <210> 24 <211> 34 <212> DNA <213> Artificial sequence <220> <223> ALSS R <400> 24 CTCTGAGAGCTTTTTTTTTTTTTTTTTTTTTTT 34 <210> 25 <211> 34 <212> DNA <213> IVD F <400> 25 CTCTGAGC TCCCTAGAGAG ctttcgtttt catg 34 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> kivD R <400> 26 ctctctctcg agttatgatt tattttgttc agc 33 <210> 27 <211> 49 <212> DNA < 213> Artificial Sequence <220> <223> ilvC F <400> 27 ctctctgagc tcaaggagat ataccatggc taactacttc aatacactg 49 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ilvC R <400> 28 ctctctgtcg acttaacccg caacagcaat ac 32 <210> 29 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ilvD F <400> 29 ctctctgtcg acaaggagat ataccatgcc taagtaccgt tcc 43 <210> 30 <2 11> 32 < 212> DNA <213> Artificial Sequence <220> <223> ilvD R <400> 30 ctctctgcgg ccgcttaacc ccccagtttc ga 32 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> pntAB F <400> 31 ctctctggat ccgatgcgaa ttggcatacc aagag 35 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> pntAB R <400> 32 ctctctgagc tcttacagag ctttcaggat tgcatc 36 <210> 3 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> bktB F <400> 33 ctctctggat ccgatggcga ccggcaaagg cgc 33 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> bktB R <400> 34 ctctctacta gttcagccca tatgcaggcc gc 32 <210> 35 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> fdh F <400> 35 ctctctcata tgatgaaaat tgttctggtg ctgtatg 37 <21 0> 36 < 211> 39 <212> DNA <213> Artificial Sequence <220> <223> fdh R <400> 36 ctctctctcg agtaattttt tatcgtgttt accgtacgc 39 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <220> < 223> yfjB(nadK) F <400> 37 ctctctggat ccgatgaata atcatttcaa gtgtattg 38 <210> 38 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> yfjB(nadK) R <400> 38 ctctctgagc tcttaga ata atttttttga ccagc 35 <210> 39 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> noxE F <400> 39 ctctctggat ccgatgaaaa tcgtagttat cgg 33 <210> 40 <211> 33 <212> DNA < 213> Artificial Sequence <220> <223> noxE R <400> 40 ctctctgagc tcttattttg catttaaagc tgc 33 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> phaB F <400> 41 GGCAGAATTC GCACTCAGC GCATTGTA TGT 33 <210> 42 <211> 29 <212> DNA <213> Artificial sequence <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> phaC F <400> 43 tacgaagctt atggcgaccg gcaaaggcgc 30 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> phaC R<400> 44 tggcggatcc tcatgccttg gctttgacg 29

Claims (11)

an isobutanol synthesis operon that is at least one selected from the group consisting of alsS, kivD, ilvC, ilvD genes and combinations thereof; a polyhydride that is at least one selected from the group consisting of bktB, phaB, phaC genes and combinations thereof A strain that simultaneously produces isobutanol and polyhydroxybutyrate, expressing polyhydroxybutyrate (PHB) synthetic operon and pntAB gene,
The strain is Escherichia coli.
delete delete delete The method of claim 1, wherein each gene consists of polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15. , strain.
delete delete i) cultivating the strain of claim 1 or 5 in a medium; and
ii) recovering isobutanol and polyhydroxybutyrate from the cultured medium and strain; A method for simultaneously producing isobutanol and polyhydroxybutyrate.
The method of claim 8, wherein the medium of step i) contains yeast extract at 0.1%v/v to 1%v/v.
The method of claim 8, wherein in step ii), isobutanol is recovered from the medium and polyhydroxybutyrate is recovered from the strain.
A composition for simultaneous production of isobutanol and polyhydroxybutyrate, comprising the strain of claim 1 or 5 and its culture medium.