KR102361618B1 - Copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate and method of preparing the same - Google Patents

Abstract

A recombinant microorganism for producing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units and a method for preparing the same, and a method for preparing a copolymer of 2-hydroxybutyrate and 3-hydroxybutyrate using the same provided

Description

Copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate and method for preparing the same

A recombinant microorganism for producing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units and a method for preparing the same, and a method for preparing a copolymer of 2-hydroxybutyrate and 3-hydroxybutyrate using the same provided

Polyhydroxyalkanoate (PHA) is a microorganism that accumulates inside microorganisms for storage of energy and reducing ability when a carbon source is abundant in a state in which the elements necessary for growth such as nitrogen, oxygen, phosphorus, and magnesium are insufficient. It is a natural polyester material. PHA is recognized as a material to replace conventional synthetic plastics because it has properties similar to synthetic polymers derived from conventional petroleum and exhibits biodegradability and biocompatibility.

There are more than 150 known monomers of PHA, and most of them are 3-, 4-, 5- or 6-hydroxyalkanoate (HA), and a representative PHA monomer that is being actively studied is 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), 3-hydroxypropionate (3HP), and medium chains with 6 to 12 carbon atoms and monomers having a hydroxyl group at the 3rd and 4th carbon positions, such as 3-hydroxyalkanoate (MCL 3-hydroxyalkanoate) of medium chain length (MCL).

The enzyme that plays a key role in synthesizing PHA in microorganisms is PHA synthetase, which uses various hydroxyacyl-CoA as a substrate to synthesize polyester containing the monomer. In addition, since the PHA synthetase has substrate specificity among various hydroxyacyl-CoA, the monomer composition of the polymer is controlled by the PHA synthetase. Therefore, in order to synthesize PHA, a metabolic pathway for synthesizing and providing various hydroxyacyl-CoA that can be used as a substrate for a PHA synthetase and a metabolic pathway for polymer synthesis using the substrate and PHA synthetase are required.

On the other hand, in the case of a monomer such as 2-hydroxybutyrate (2HB) having a hydroxyl group at the 2nd carbon position, there is a problem in that it is not suitable for the substrate specificity of the PHA synthetase.

KR 10-1211767 B1

The present invention suppresses lactate production by microorganisms by inhibiting lactate production from pyruvate and/or enhancing pyruvate production from lactate during the metabolic process of microorganisms, thereby providing 2-hydroxybutyrate and 3-hydroxy A technique for synthesizing a copolymer comprising butyrate is provided.

One example provides a recombinant microorganism engineered such that a gene encoding an enzyme for producing lactate from pyruvate is inactivated and/or a gene encoding an enzyme for producing pyruvate from lactate is overexpressed.

Another example provides a composition for preparing a copolymer comprising the recombinant microorganism, 2-hydroxybutyrate and 3-hydroxybutyrate.

Another example provides a method for preparing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate, comprising culturing the recombinant microorganism. The culturing step may be performed in a medium supplied with 2-hydroxybutyrate and 3-hydroxybutyrate as monomers.

Another example provides a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate. The copolymer may be prepared by the manufacturing method described above.

In order to biosynthesize the 2-hydroxybutyrate-3-hydroxybutyrate copolymer [P(2HB-3HB)], the lactate production ability is inhibited in order to inhibit the use of lactate produced during microbial fermentation as a monomer of the copolymer. Recombinant strains are required.

The present invention suppresses lactate production by microorganisms by inhibiting lactate production from pyruvate and/or enhancing pyruvate production from lactate during the metabolic process of microorganisms, thereby providing 2-hydroxybutyrate and 3-hydroxy It is characterized by providing a technique for synthesizing a copolymer of oxybutyrate.

One example provides a recombinant microorganism engineered such that a gene encoding an enzyme for producing lactate from pyruvate is inactivated, and/or a gene encoding an enzyme for producing pyruvate from lactate is overexpressed. The recombinant microorganism may include an endogenous and/or exogenous (homologous or heterologous) CoA transferase-encoding gene and a PHA synthetase-encoding gene.

The recombinant microorganism may be used to prepare a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units.

Another example provides a composition for preparing a copolymer comprising the recombinant microorganism, 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units.

Another example provides a method for preparing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate, comprising culturing the recombinant microorganism. The culturing step may be performed in a medium supplied with 2-hydroxybutyrate and/or a precursor thereof as a monomer, and 3-hydroxybutyrate and/or a precursor thereof. The medium may be lactate-free.

Another example provides a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units. The copolymer may be prepared by the above-described manufacturing method. The copolymer has a lactate content of about 10 mol% or less (the lower limit includes 0 mol%, hereinafter the same), about 8 mol% or less, about 7 mol% or less, about 6 mol% or less, about 5 mol% or less , about 4 mole % or less, about 3 mole % or less, about 2 mole % or less, about 1 mole % or less, or about 0 mole %.

As used herein, the term "copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units" or "2-hydroxybutyrate-3-hydroxybutyrate copolymer" refers to a repeating unit monomer. As, 2-hydroxybutyrate and 3-hydroxybutyrate refer to a linear polyester including repeating units polymerized by ester bonds. At this time, the polymerization sequence of each monomer is not particularly limited, and may be randomly repeated.

The 2-hydroxybutyrate-3-hydroxybutyrate copolymer [P(2HB-3HB)] is coenzyme-A (CoA) that transfers coenzyme-A (CoA) to 2-hydroxybutyrate and 3-hydroxybutyrate monomers. ) can be biosynthesized by PHA synthetase that synthesizes transferase and polyhydroxyalkanoate (PHA).

The recombinant microorganism provided herein may include a CoA transferase-encoding gene and a PHA synthetase-encoding gene, for the production of a 2-hydroxybutyrate-3-hydroxybutyrate copolymer, the CoA transferase and PHA The synthetase is the same as described above. Genes encoding the CoA transferase and PHA synthetase are introduced into microbial cells by a conventional genetic recombination method (eg, using a recombinant vector comprising a CoA transferase-encoding gene and a PHA synthetase-encoding gene, respectively or together). there may be

In addition, in the recombinant microorganism, in order to increase the production efficiency of the 2-hydroxybutyrate-3-hydroxybutyrate copolymer, a gene encoding an enzyme for producing lactate from pyruvate is inactivated and/or pyruvate from lactate It may be engineered to overexpress a gene encoding an enzyme that produces a bait. The inactivation of the gene encoding the enzyme for producing lactate from pyruvate is performed by deleting (removing) all or part of the gene encoding the enzyme for producing lactate from pyruvate in the genome of a recombinant microorganism in a conventional manner. can be The overexpression of a gene encoding an enzyme for producing pyruvate from lactate is performed by using a recombinant vector comprising a gene encoding an enzyme for producing pyruvate from lactate derived from the same individual, allogeneic, or heterologous to the recombinant microorganism. It can be carried out by a conventional genetic recombination method used.

In the recombinant microorganism, the CoA transferase-encoding gene, the PHA synthetase-encoding gene, and the gene encoding the enzyme for producing pyruvate from lactate are each included (inserted) in separate recombinant vectors, or two or more of them Together they can be included (inserted) into one vector and introduced into the microorganism.

The CoA transferase may be, for example, propionyl-CoA transferase. Propionyl-CoA transferase (EC 2.8.3.1) is an enzyme that catalyzes the chemical reaction of Scheme 1:

acetyl-CoA + propanoate ⇔ acetate + propanoyl-CoA (Scheme 1).

The enzyme and the gene encoding it may be derived from Clostridium propionicum .

For example, the propionyl-CoA transferase encoding gene is

(a) the nucleotide sequence of SEQ ID NO: 1;

(b) a nucleotide sequence including a mutation in the nucleotide sequence of SEQ ID NO: 1 A1200G (meaning a mutation in which A, which is the 1200th base, is substituted with G; the same applies to the expression of nucleotide sequence mutations described below);

(c) a nucleotide sequence including mutations of T78C, T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1;

(d) contains the mutation of A1200G in the nucleotide sequence of SEQ ID NO: 1, and in the amino acid sequence corresponding to SEQ ID NO: 1, G335A (meaning the mutation in which the 335th amino acid Gly is substituted with Ala, in the amino acid sequence variation described below a nucleotide sequence encoding an amino acid sequence including a mutation of

(e) a nucleotide sequence encoding an amino acid sequence containing the mutation of A1200G in the nucleotide sequence of SEQ ID NO: 1 and including the mutation of A243T in the amino acid sequence corresponding to SEQ ID NO: 1;

(f) a nucleotide sequence encoding an amino acid sequence containing mutations of T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1 and including a mutation of D65G in the amino acid sequence corresponding to SEQ ID NO: 1;

(g) a nucleotide sequence encoding an amino acid sequence containing the mutation of A1200G in the nucleotide sequence of SEQ ID NO: 1 and including the mutation of D257N in the amino acid sequence corresponding to SEQ ID NO: 1;

(h) a nucleotide sequence encoding an amino acid sequence containing mutations of T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1 and including a mutation of D65N in the amino acid sequence corresponding to SEQ ID NO: 1;

(i) a nucleotide sequence encoding an amino acid sequence comprising mutations of T669C, A1125G, and T1158C in the nucleotide sequence of SEQ ID NO: 1, and including a mutation of T199I in the amino acid sequence corresponding to SEQ ID NO: 1; and

(j) a nucleotide sequence encoding an amino acid sequence containing mutations of T78C, T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1, and including a mutation of V193A in the amino acid sequence corresponding to SEQ ID NO: 1

It may include a nucleotide sequence selected from the group consisting of, and the propionyl-CoA transferase may include an amino acid sequence encoded by the nucleotide sequence.

The polyhydroxyalkanoate synthase (polyhydroxyalkanoate (PHA) synthase) is an enzyme that biosynthesizes polyhydroxyalkanoate using CoA and a thioester of hydroxy fatty acid as a substrate, and uses a fatty acid having 3-5 carbon atoms. type (e.g. Cupriavidus necator , Alcaligenes latus , etc.) and a type using fatty acids having 6-14 carbon atoms (eg, derived from the genus Pseudomonas ).

For example, the PHA synthetase and the gene encoding it may be a PHA synthetase (phaC) derived from a Pseudomonas sp. strain, for example, a Pseudomonas sp. 6-19 strain.

For example, the PHA synthetase is

the amino acid sequence of SEQ ID NO: 4; or

In the amino acid sequence of SEQ ID NO: 4, comprising at least one mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R and A527S amino acid sequence

may include.

In another embodiment, the PHA synthetase is

In the amino acid sequence of SEQ ID NO: 4,

(i) S325T and Q481M;

(ii) E130D, S325T and Q481M;

(iii) E130D, S325T, S477R and Q481M;

(iv) E130D, S477F and Q481K; and

(v) L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K and A527S

It may include an amino acid sequence containing a mutation selected from the group consisting of, and the PHA synthetase encoding gene may include a nucleotide sequence encoding the amino acid sequence.

The enzyme for generating pyruvate from lactate is D-lactate dehydrogenase, which is an aerobic lactate dehydrogenase derived from the same individual (strain) or a microorganism homogeneous or heterogeneous as the recombinant microorganism (recombinant host microorganism). (D-lactate dehydrogenase; Dld; EC 1.1.1.28), for example, may be D-lactate dehydrogenase derived from a microorganism homologous to the recombinant microorganism (eg, E. coli). In one embodiment, the D-lactate dehydrogenase is E. coli (eg, Escherichia coli D-lactate dehydrogenase (eg, GenBank Accession No. NP_416637.1 (coding gene: Genbank ID 946653; SEQ ID NO: 38)) from K-12 MG1655 strain).

The enzyme for producing lactate from pyruvate may be a lactate dehydrogenase (Ldh) native to the recombinant microorganism, for example, lactate dehydrogenase A (LdhA).

The enzymes may include additional mutations within a range that does not entirely alter the activity of the molecule. For example, amino acid exchanges in proteins and peptides that do not entirely alter the activity of the molecule are known in the art. For example, exchanges that normally occur are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe , Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, the protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like. In addition, it may include an enzyme protein in which the structural stability to heat, pH, etc. of the protein is increased or the protein activity is increased due to mutation or modification in the amino acid sequence.

In addition, the gene encoding the enzyme may include a nucleic acid molecule comprising a functionally equivalent codon or a codon encoding the same amino acid (by codon degeneracy), or a codon encoding a biologically equivalent amino acid. have. The nucleic acid molecule may be isolated or prepared using standard molecular biology techniques, for example, chemical synthesis or recombinant methods, or commercially available ones.

"Vector" refers to a genetic construct comprising essential regulatory elements operably linked to express a gene insert encoding a target protein in a cell of an individual, and means for introducing a nucleic acid sequence encoding a target protein into a host cell becomes this The vector may be one or more selected from the group consisting of various vectors such as plasmids, adenoviral vectors, retroviral vectors and adeno-associated viral vectors such as viral vectors, bacteriophage vectors, cosmid vectors, YAC (Yeast Artificial Chromosome) vectors, etc. have. In one embodiment, the plasmid vector is pBlue (eg, pBluescript II KS(+)), pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, It may be at least one selected from the group consisting of pGEX series, pET series, pUC19, etc., and the bacteriophage vector may be at least one selected from the group consisting of lambda gt4 lambda B, lambda-Charon, lambda Δz1, and M13, etc. The viral vector may be SV40, but is not limited thereto.

"Recombinant vector" includes cloning vectors and expression vectors containing an exogenous target gene. A cloning vector is a replicon that includes an origin of replication, for example, a replication origin of a plasmid, phage or cosmid, to which other DNA fragments are attached and the attached fragment can be replicated. Expression vectors have been developed for use in synthesizing proteins.

In the present specification, the vector is not particularly limited as long as it can express the desired enzyme gene in various host cells such as prokaryotic or eukaryotic cells and function to produce it. It is recommended that the fusion be reversed so that the gene expression in the cell can be stably maintained for a long period of time.

Such vectors contain transcriptional and translational expression control sequences that allow the gene of interest to be expressed in the selected host. Expression control sequences may include a promoter for effecting transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and/or sequences regulating the termination of transcription and translation. . For example, regulatory sequences suitable for prokaryotes may include a promoter, optionally an operator sequence, and/or a ribosome binding site. Regulatory sequences suitable for eukaryotes may include promoters, terminators and/or polyadenylation signals. The start codon and stop codon are generally considered part of the nucleic acid sequence encoding the protein of interest, and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. Also, in the case of an expression vector capable of replication, the origin of replication may be included. In addition, enhancers, untranslated regions at the 5' and 3' ends of the gene of interest, selection markers (eg, antibiotic resistance markers), or replication competent units may be included as appropriate. Vectors may be self-replicating or integrated into host genomic DNA.

Examples of useful expression control sequences include the early and late promoters of adenovirus, simian virus 40 (SV40), the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalo. virus (CMV) promoter, Epstein virus (EBV) promoter, Loose sarcoma virus (RSV) promoter, RNA polymerase II promoter, β-actin promoter, human hegglobin promoter and human muscle creatine promoter, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter region of phage lambda, regulatory region of fd coding protein, promoter for phosphoglycerate kinase (PGK) or other glycolytic enzymes, promoter for phosphatase for example, Pho5, promoters of yeast alpha-crossing systems and other sequences of construction and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof. .

In order to increase the expression level of a transgene in a cell, a desired gene and transcriptional and translational expression control sequences must be operably linked to each other. In general, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, in contact and in reading frame. For example, DNA for a pre-sequence or secretion leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the protein, and a promoter or enhancer is The ribosome binding site may be operably linked to a coding sequence if it affects the transcription of the sequence, or the ribosome binding site may be operably linked to a coding sequence if it affects the transcription of the sequence, or the ribosome binding site may be operably linked to a coding sequence to facilitate translation. It can be operably linked to a coding sequence when configured to do so. Linking of these sequences can be performed by ligation (ligation) at convenient restriction enzyme sites, and when such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used. can be performed.

Those skilled in the art will take into account the properties of the host cell, the number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, for example, an antibiotic marker, various vectors suitable for the present invention, expression control Sequence, host, etc. can be selected.

The recombinant microorganism provided herein can be obtained by transforming a host microbial cell using the recombinant vector.

The term "transformation" means that a target gene is introduced into a host microorganism so that the phloem gene becomes replicable as an extrachromosomal factor or by chromosomal integrity completion.

Microorganisms usable as a host microorganism may be selected from the group consisting of prokaryotic cells and eukaryotic cells, and typically, a microorganism having a high DNA introduction efficiency and a high DNA expression efficiency may be used as the host microorganism. Host microorganisms include, in embodiments, E. coli (eg, E. coli DH5a, E. coli JM101 , E. coli K12, E. coli W3110, E. coli X1776, E. coli B and E. coli XL1-Blue) including Escherichia, Pseudomonas, Bacillus, Streptomyces, Erwinia, Serratia, Providencia, Corynebacterium, Leptospira, Salmonella, Brevi Bacterial genus, Hypomonas genus, Chromobacterium genus, Nocadia genus, and well-known eukaryotic and prokaryotic hosts such as fungi or yeast may be exemplified, but the present invention is not limited thereto. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself.

In addition, for the purposes of the present invention, the host cell may be a microorganism having a pathway for biosynthesis of hydroxyacyl-CoA from a carbon source.

Transformation methods include suitable standard techniques as known in the art, for example, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation. precipitation), calcium chloride/rubidium chloride method, retroviral infection, DEAE-dextran, cationic liposome method, polyethylene glycol-mediated uptake, gene gun (gene gun) may be used, but is not limited thereto. At this time, the circular vector can be digested with an appropriate restriction enzyme and introduced in the form of a linear vector.

By culturing the transformed recombinant microorganism, 2-hydroxybutyrate-3-hydroxybutyrate copolymer may be produced.

By culturing the recombinant microorganism into which the recombinant vector is introduced in an appropriate medium, 2-hydroxybutyrate-3-hydroxybutyrate copolymer can be effectively produced. The medium and culture conditions used at this time may be appropriately selected and used according to the type of recombinant microorganism. In culture, conditions such as temperature, pH of the medium, and incubation time can be appropriately adjusted so as to be suitable for cell growth and production of the copolymer. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch culture.

In one embodiment, the culture may be performed in a medium containing 2-hydroxybutyrate or a precursor thereof, and 3-hydroxybutyrate or a precursor thereof. The precursor of 2-hydroxybutyrate and/or 3-hydroxybutyrate may be any substance convertible to 2-hydroxybutyrate and/or 3-hydroxybutyrate during the metabolic process of microorganisms, for example, ketobutyrate, glucose etc. can be exemplified. In addition, if the recombinant microorganism can biosynthesize 2-hydroxybutyrate and 3-hydroxybutyrate from a carbon source such as glucose, 2-hydroxybutyrate, 3-hydroxybutyrate, and/or their precursors are not separately added. The copolymer can be prepared without a conventional carbon source supply.

In addition, the medium used for culturing should suitably satisfy the requirements of a specific strain. The medium may include various carbon sources, nitrogen sources, phosphorus and trace elements. Carbon sources in the medium include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, and linoleic acid At least one selected from the group consisting of fatty acids, glycerol, alcohols such as ethanol, and organic acids such as acetic acid may be exemplified, but the present invention is not limited thereto. These substances may be used individually or as a mixture. Nitrogen sources in the medium include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, However, the present invention is not limited thereto. The nitrogen sources can also be used individually or as a mixture. As the phosphorus in the medium, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or a corresponding sodium-containing salt may be exemplified, but not limited thereto. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth, or may contain essential growth materials such as amino acids and vitamins, but is not limited thereto. The above-mentioned raw materials may be added batchwise or continuously by a method suitable for the culture during the culturing process.

In addition, if necessary, a basic compound such as sodium hydroxide, potassium hydroxide, ammonia, or an acid compound such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture. In addition, an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles. Oxygen or oxygen-containing gas (eg, air) may be injected into the culture to maintain aerobic conditions, and the temperature of the culture may be usually 20°C to 45°C, preferably 25°C to 40°C. Cultivation can be continued until the desired polymer yield is maximized.

In the method for producing a 2-hydroxybutyrate-3-hydroxybutyrate copolymer provided in the present invention, after culturing the recombinant microorganism, the produced 2-hydroxybutyrate-3-hydroxybutyrate copolymer is obtained from the culture. It may further comprise the step of recovery (or separation or purification).

The 2-hydroxybutyrate-3-hydroxybutyrate copolymer produced from a recombinant microorganism can be isolated from cells or culture media by methods well known in the art. Examples of the recovery method of 2-hydroxybutyrate-3-hydroxybutyrate copolymer include centrifugation, sonication, filtration, ion exchange chromatography, high performance liquid chromatography (HPLC), gas chromatography ( gas chromatography: GC), but is not limited to these examples.

The present invention provides a technology capable of efficiently producing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate, and the produced 2-hydroxybutyrate-3-hydroxybutyrate copolymer is biodegradable And as a raw material of bioplastic with biocompatibility, it can be widely used in electronics, automobiles, food, agriculture and medical fields.

1 shows the construction process and cleavage map of the pPs619C1310-CpPCT540 vector.
2 shows a cleavage map of the pPs619C1249.18H-CPPCT540 vector.
3 shows a cleavage map of the pCDFJ23- dld vector.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. 2- hydroxybutyrate -3- hydroxybutyrate Preparation of Recombinant Vector for Copolymer Preparation

1-1. Preparation of pPs619C1310-CPPCT540 recombinant vector

The propionyl-CoA transferase gene (pct) was used as a variant of propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum, and the PHA synthetase gene was Pseudomonas genus MBEL A variant of the PHA synthetase derived from 6-19 (KCTC 11027BP) was used. The vector used at this time is pBluescript II (Stratagene Co., USA).

First, in order to isolate the PHA synthetase (phaC1Ps6-19) gene, the entire DNA of Pseudomonas genus MBEL 6-19 (KCTC 11027BP) was extracted, and based on the phaC1Ps6-19 gene sequence (SEQ ID NO: 3), primer [5 '-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3' (SEQ ID NO: 5), 5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3' (SEQ ID NO: 6)] was prepared, PCR was performed using the extracted total DNA as a template. By electrophoresis of the obtained PCR product, a 1.7 kb gene fragment corresponding to the phaC1Ps6-19 gene was identified, and the phaC1Ps6-19 gene was obtained.

In order to express the phaC1Ps6-19 synthetase, the DNA fragment containing the PHB-producing operon derived from Ralstonia eutropha H16 in the pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347) was used with BamHI/EcoRI. It was cut and inserted into the BamHI/EcoRI recognition site of pBluescript II (Stratagene Co., USA) to prepare a pReCAB recombinant vector. In the pReCAB vector, PHA synthetase (phaCRE) and monomer supply enzymes (phaARE and phaBRE) are constitutively expressed by the PHB operon promoter. In order to construct a phaC1Ps6-19 synthetase gene fragment containing only one BstBI/SbfI recognition site at each end, the BstBI site was first removed without amino acid conversion by SDM (site directed mutagenesis) method, and the BstBI/SbfI recognition site Primer [5'-ATG CCC GGA GCC GGT TCG AA -3' (SEQ ID NO: 7), 5'-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC - 3' (SEQ ID NO: 8), 5 to add '- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3' (SEQ ID NO: 9), 5- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC 3' (SEQ ID NO: 10), 5'-GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG 3' (SEQ ID NO: 11), 5'-aac ggg agg gaa cct gca gg -3' (SEQ ID NO: 12)] was used to perform overlapping PCR. The pReCAB vector was digested with BstBI/Sbfl to remove R. eutropha H16 PHA synthetase (phaCRE), and then the phaC1Ps6-19 gene obtained above was inserted into the BstBI/Sbfl recognition site to prepare a pPs619C1-ReAB recombinant vector.

Three amino acid positions affecting SCL (short chain length) activity were found through amino acid sequence analysis, and primers [5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC-3' (SEQ ID NO: 13), 5 - GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG-3' (SEQ ID NO: 14), 5'-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC-3' (SEQ ID NO: 15), 5'- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG-3' (SEQ ID NO: 16), 5'-ATC AAC CTC ATG ACC GAT GCG ATG GCG CCG ACC-3' (SEQ ID NO: 17), 5'- GGT CGG CGC CAT CGC ATC GGT CAT GAG GTT GAT-3' (SEQ ID NO: 18)] using the SDM method to prepare pPs619C1300-ReAB containing phaC1Ps6-19300, which is a phaC1Ps6-19 synthetase variant including E130D, S325T, and Q481M did

Here, propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum was used to construct a constitutively expressed system in the form of an operon in which propionyl-CoA transferase is expressed together. was used. CP-PCT is Clostridium propionicum chromosomal DNA primer [5'-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3' (SEQ ID NO: 19), 5'-GC TCTAGA TTA GGA CTT CAT TTC CTT CAG ACC CAT TAA GCC TTC TG-3' (SEQ ID NO: 20)], a fragment obtained by PCR was used. At this time, the NdeI site originally present in the wild-type CP-PCT was removed using the SDM method for ease of cloning, and a primer [5'-AGG CCT GCA GGC GGA TAA CAA TTT CAC was used to add the SbfI/NdeI recognition site. ACA GG-3' (SEQ ID NO: 21), 5'-GCC CAT ATG TCT AGA TTA GGA CTT CAT TTC C-3' (SEQ ID NO: 22)] was used to perform overlapping PCR. The PPS619C1300-REAB vector was digested with SBFI/NdeI to remove Ralstonia eutrophus H16-derived monomer supply enzymes (phaARE and phaBRE), and then the PCR-cloned CP-PCT gene was inserted into the SbfI/NdeI recognition site to insert the pPs619C1300-CPPCT recombinant vector. was prepared.

Next, using the pPs619C1300-CPPCT prepared above as a template to introduce random mutagenesis into the CP-PCT gene, primers [5'-CGCCGGCAGGCCTGCAGG-3' (SEQ ID NO: 23), 5'-GGCAGGTCAGCCCATATGTC -3' (SEQ ID NO: 24)], Mn2+ was added and Error-prone PCR was performed under conditions where there was a difference in the concentration of dNTPs. Then, PCR was performed under normal conditions using the primers to amplify the PCR fragment containing the random mutation. After the pPs619C1300-CPPCT vector was digested with SbfI/NdeI to remove wild-type CP-PCT, the amplified mutant PCR fragment was inserted into the SbfI/NdeI recognition site to make a ligation mixture, introduced into E. coli JM109, and introduced into ~105 scale. A CP-PCT library was prepared. The prepared CP-PCT library was grown for 3 days in a polymer detection medium (LB agar, glucose 20g/L, 3HB 1g/L, Nile red 0.5μg/ml), and then a screening operation was performed to check whether the polymer was generated. About 80 candidates were first selected. These candidates were cultured in liquid for 4 days (LB agar, glucose 20g/L, 3HB 1g/L, ampicillin 100mg/L, 37°C) under the conditions in which polymers were produced, and through FACS (Florescence Activated Cell Sorting) analysis, 2 individuals, That is, CP-PCT Variant 512 (including nucleic acid substitution A1200G) and CP-PCT Variant 522 (including nucleic acid substitution T78C, T669C, A1125G, and T1158C) were selected. Various CP-PCT variants could be obtained by performing random mutations again by the Error-prone PCR method based on the primary selected mutants (CP-PCT Variant 512, CP-PCT Variant 522). CP-PCT Variant 540 (including Val193Ala and silent mutations T78C, T669C, A1125G, T1158C) was secondarily selected to prepare a pPs619C1300-CPPCT540 vector.

In addition, based on the phaC1Ps6-19 synthetase variant (phaC1Ps6-19300) prepared above, primers [5'-GAA TTC GTG CTG TCG AGC CGC GGG CAT ATC-3' (SEQ ID NO: 25), 5'-GAT ATG CCC GCG GCT CGA CAG CAC GAA TTC-3' (SEQ ID NO: 26), 5'-GGG CAT ATC AAG AGC ATC CTG AAC CCG C-3' (SEQ ID NO: 27), 5'-G CGG GTT CAG GAT GCT CTT GAT ATG CCC-3' (SEQ ID NO: 28)] using the SDM method using E130D, S477F and Q481K pPs619C1310- containing PHA synthetase variant (phaC1Ps6-19310) derived from MBEL 6-19 of Pseudomonas genus having a mutated amino acid sequence CPPCT540 vector was prepared (FIG. 1).

1-2. Preparation of pPs619C1249.18H-CPPCT540 recombinant vector

Using the pPs619C1310-CPPCT540 vector prepared in 1-1 above as a template, error-prone PCR using primers [5'-ATGCCCGGAGCCGGTTCGAA-3' (SEQ ID NO: 29) and 5'-GAAATTGTTATCCGCCTGCAGG-3' (SEQ ID NO: 30)] was performed. After performing error-prone PCR, PCR was performed again using the primers to amplify the PCR fragment containing the mutation, and then the amplified mutations were inserted into the BstBI/SbfI position of the pPs619C1310-CPPCT540 vector to prepare a library of mutants. did The prepared mutant library was transformed into E.coli XL-1Blue and cultured for 3 days in PHB detection medium (LB agar, glucose 20g/L, Nile red 0.5μg/ml). The mutant finally selected through the screening process after culture was pPs619C1249.18H having an amino acid sequence in which L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K and A527S were mutated. In this way, a recombinant vector pPs619C1249.18H-CPPCT540 vector was prepared (FIG. 2).

Example 2. 2- hydroxybutyrate -3- hydroxybutyrate Preparation of Recombinant Strain for Copolymer Preparation

2.1. ldhA Gene knockout (knock-out) mutant production

Escherichia Escherichia involved in lactate production during the metabolic process of E. coli to produce a lactate-free polymer based on coli XL1-Blue (Stratagene, USA) coli XL1-blue-derived D-lactate dehydrogenase gene ( ldhA ) was knocked out from genomic DNA, and ldhA -deleted E. coli mutant E. coli XL1-Blue ( ΔldhA ) was prepared. Gene deletion was performed using a red-recombination method well known in the art. The oligomer used to delete ldhA was synthesized with the nucleotide sequence of SEQ ID NO: 31 (5'-ATCAGCGTACCCGTGATGCTAACTTCTCTCTGGAAGGTCTGACCGGCTTTAATTAACCCTCACTAAAGGGCG-3') and SEQ ID NO: 32 (5'-ATCAGCGTACCCGTGATGCTAACTTCTCTCTCTGGAAGGTCTGACCGGCTTTAATTAACCC-3').

2.2. dld Gene overexpression mutant production

E. coli XL1-blue (Stratagene, USA) and the ldhA deletion E. coli mutant E. coli prepared in Example 2.1 XL1-Blue ( ΔldhA ) to E. coli , respectively XL1-blue-derived Aerobic lactate dehydrogenase (D-lactate dehydrogenase) gene ( dld ) (SEQ ID NO: 38) By introducing a recombinant vector pCDFJ23- dld (see FIG. 3) containing the, dld overexpressed E. coli mutant E. coli XL1-Blue ( + dld ) and E. coli XL1-Blue ( ΔldhA :: dld ) were prepared, respectively.

In more detail, first, a pCDFJ23 vector (2.3kb) was constructed to express the Dld enzyme. In the pCDFH23 vector, a DNA fragment containing two T7 promoters was cut with XbaI/XhoI in pCDFDuet ^TM -1 (Novagen Co., USA, 3.7kb), and constitutively expressed J23101 and J23108 promoters were placed at the XbaI/XhoI recognition site. inserted. The size of the inserted DNA fragment is 328 bp (SEQ ID NO: 33). For J23101 and J23108 promoter insertion, XbaI / XhoI recognition site added primer [5'-TAC TGA ACC GCT CTA GAT TTA CAG CTA GC -3' (SEQ ID NO: 34), 5'-CTT TAC CAG ACT CGA GTT CGA AGA CGT CA-3' (SEQ ID NO: 35)] was used.

Aerobic lactate dehydrogenase (D-lactate dehydrogenase) gene ( dld ) gene was inserted into the NcoI/SbfI recognition site of the prepared CDFJ23 vector. Aerobic lactate dehydrogenase (D-lactate dehydrogenase) gene ( dld ) To isolate the gene, the total DNA of E. coli K-12 MG1655 was extracted, and based on the dld gene sequence (Genbank ID 946653; SEQ ID NO: 38), NcoI Primer containing /Sbfl recognition site [5'-TCT GAC CCA TGG ATG TCT TCC ATG ACA ACA-3' (SEQ ID NO: 36), 5'-TGT CGA CCT GCA GGT TAC TCC ACT TCC TGC CA-3' (SEQ ID NO: 36) No. 37)], and PCR was performed using the extracted total DNA as a template. The obtained PCR product was electrophoresed to confirm a 1.7 kb gene fragment corresponding to the dld gene, and the dld gene was obtained. By inserting the dld gene into the pCDFJ23 vector, pCDFJ23- dld (4.1kb; see FIG. 3) was prepared.

2.3. 2- hydroxybutyrate -3- hydroxybutyrate Recombinant strain production for copolymer production

E. coli XL1-blue (Stratagene, USA), ldhA -deleted E. coli mutant E. coli XL1-Blue ( ΔldhA ) prepared in Example 2.1, and E. coli mutant E. coli XL1-Blue (+ dld ) prepared in Example 2.2 ) and E. coli XL1-Blue ( ΔldhA :: dld ) were transformed by electroporation using the recombinant vector pPs619C1249.18H-CPPCT540 prepared in Example 1.2, respectively, to prepare 2HB-3HB copolymer A recombinant strain was prepared.

Example 3. Preparation of 2-hydroxybutyrate-3-hydroxybutyrate copolymer

3 mL of LB medium [Bacto ^TM Triptone (BD) 10 g / L, Bacto ^TM yeast extract (BD) 5 g / L, NaCL ( amresco) 10 g/L] for 12 hours prior to incubation (seed culture).

100 ml of the obtained pre-culture solution was mixed with 100 mg/L of ampicillin, 2-hydroxybutyrate (2HB) 0.5 g/L or 1 g/L, 3-hydroxybutyrate (3HB) 0.5 g/L or 1 g/L, glucose 20 g/L and 100 ml MR medium additionally containing 10 mg/L of thiamine (Glucose 10 g, KH ₂ PO ₄ 6.67 g, (NH ₄ )2HPO _{4 4} g, MgSO ₄ .7H ₂ O 0.8 g, citric acid 0.8 g per 1 L of medium, and trace metal solution 5mL; Here, trace metal solution is 5mL of 5M HCl per 1L, FeSO ₄ 7H ₂ O 10 g, CaCl _{2 2} g, ZnSO ₄ 7H ₂ O 2.2 g, MnSO ₄ 4H ₂ O 0.5 g, CuSO ₄ 1 g 5H ₂ O, (NH ₄ )6Mo ₇ O ₂ 4H ₂ O 0.1 g, and Na ₂ B ₄ O ₂ 10H ₂ O containing 0.02 g), the main culture was performed while stirring at 30 ° C. at 250 rpm for 4 days.

The culture solution was centrifuged at 4°C and 4000 rpm for 10 minutes to recover the cells, washed twice with a sufficient amount of distilled water, and then dried at 80°C for 12 hours. After quantifying the removed cells, chloroform was used as a solvent at 100° C. and reacted with methanol under a sulfuric acid catalyst. At room temperature, distilled water in a volume equivalent to half that of chloroform was added and mixed, and then left to stand until separated into two layers. Among the two layers, the chloroform layer in which the methylated polymer monomers were dissolved was collected, and the polymer composition was analyzed by gas chromatography (GC). As an internal standard, benzoate was used. The GC analysis conditions used at this time are shown in Table 1 below.

GC analysis conditions Item Quality Model Hewlett Packard 6890N Detector Flame ionization detector (FID) Column Alltech Capillary AT ^TM -WAX, 30m, 0.53mm Liquid phase 100% polyethylene glycol Inj.port temp/Det.port temp 250℃/ 250℃ carrier gas He Total flow 3ml/min septum purge went flow 1ml/min column head pressure 29 kPa Injection port mode Splitless Injection volume/Solvent 1 μl/chloroform Initial temp./Time 80℃/5min Final temp./Time 230℃/5min Ramp of temp. 7.5℃/min

The results obtained in the GC analysis are shown in Tables 2 and 3 below:

2HB (0.5 g/L) + 3HB (0.5 g/L) lactate mol% 2HB mol% 3HB mol% PHA content % (w/w) E. coli XL1-Blue + dld 3.3 ± 0.3 3.2 ± 0.2 93.5 ±0.4 6.0 ±0.9 - 72.2 ± 0.5 14.5 ± 0.1 13.3 ±0.5 42.1 ± 0.7 E. coli XL1-Blue ( ΔldhA ) + dld 0 3.6 ± 0.3 96.4 ± 0.3 6.5 ± 1.6 - 4.6 ± 1.0 31.4 ± 0.2 64.0 ± 0.9 12.4 ± 0.4

2HB (1 g/L) + 3HB (0.5 g/L) lactate mol% 2HB mol% 3HB mol% PHA content% (w/w) E. coli XL1-Blue + dld 6.2 ± 0.4 9.0 ± 0.4 84.8 ± 03 13.9 ± 0.3 - 61.9 ± 0.5 25.0 ± 0.4 13.1 ± 0.3 50.0 ± 1.3 E. coli XL1-Blue ( ΔldhA ) + dld 0 8.3 ± 0.9 91.7 ± 0.9 13.4 ± 1.0 - 5.5 ± 0.2 50.8 ± 0 43.6 ± 0.2 22.3 ± 0.6

(PHA content% (w/w): polymer (total content of P(2HB-3HB) and P(LA-2HB-3HB) in total cell dry weight)

As shown in Tables 2 and 3, the polymer produced from the E. coli XL1-Blue (+dld) strain overexpressing Dld in the wild-type E. coli XL1-Blue strain had a lactate content within 6 mol%, and the control E. coli XL1-Blue strain. coli XL1-Blue (-) was reduced by more than 10 times. In addition, it was confirmed that P(2HB-3HB) having a lactate content of 0 mol% was produced from the recombinant strain ( E. coli XL1-Blue ( ldhA ) + dld) overexpressing dld after removing ldhA.

<110> LG CHEM, LTD. <120> Copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate and method of preparing the same <130> DPP20174034KR <160> 38 <170> KopatentIn 3.0 <210> 1 <211> 1575 <212> DNA <213> Clostridium propionicum <220 > <221> gene <222> (1) .. (1575) <223> popionyl-CoA transferase <400> 1 atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60 acagttacaa caagtggttt cgttggaaat gcaatccctg aggctcttga tagagctgta 120 gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta cctatgttta ttgtggttct 180 caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240 tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300 atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360 cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420 ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480 caggaatatt tattctaccc tgcttttcct attcatgtag ctcttattcg tggtacttac 540 gctgatgaaa gcggaaatat cacatttgag aaagaagttg ctcctctgga aggaacttca 600 gtatgccagg ctgttaa aaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140 tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttattgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgtta tg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cctaa 1575 <210> 2 <PT211> 524 <212> PEPT. <223> propionyl-CoA transferase <400> 2 Met Arg Lys Val Pro Ile Ile Thr Ala Asp Glu Ala Ala Lys Leu Ile 1 5 10 15 Lys Asp Gly Asp Thr Val Thr Thr Ser Gly Phe Val Gly Asn Ala Ile 20 25 30 Pro Glu Ala Leu Asp Arg Ala Val Glu Lys Arg Phe Leu Glu Thr Gly 35 40 45 Glu Pro Lys Asn Ile Thr Tyr Val Tyr Cys Gly Ser Gln Gly Asn Arg 50 55 60 Asp Gly Arg Gly Ala Glu His Phe Ala His Glu Gly Leu Leu Lys Arg 65 70 75 80 Tyr Ile Ala Gly His Trp Ala Thr Val Pro Ala Leu Gly Lys Met Ala 85 90 95 Met Glu Asn Lys Met Glu Ala Tyr Asn Val Ser Gln Gly Ala Leu Cys 100 105 110 His Leu Phe Arg Asp Ile Ala Ser His Lys Pro Gly Val Phe Thr Lys 115 120 125 Val Gly Ile Gly Thr Phe Ile Asp Pro Arg Asn Gly Gly Gly Lys Val 130 135 140 Asn Asp Ile Thr Lys Glu Asp Ile Val Glu Leu Val Glu Ile Lys Gly 145 150 155 160 Gln Glu Tyr Leu Phe Tyr Pro Ala Phe Pro Ile His Val Ala Leu Ile 165 170 175 Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Phe Glu Lys Glu 180 185 190 Val Ala Pro Leu Glu Gly Thr Ser Val Cys Gln Ala Val Lys Asn Ser 195 200 205 Gly Gly Ile Val Val Val Gln Val Glu Arg Val Val Val Lys Ala Gly Thr 210 215 220 Leu Asp Pro Arg His Val Lys Val Pro Gly Ile Tyr Val Asp Tyr Val 225 230 235 240 Val Val Ala Asp Pro Glu Asp His Gln Gln Ser Leu Asp Cys Glu Tyr 245 250 255 Asp Pro Ala Leu Ser Gly Glu His Arg Arg Pro Glu Val Val Gly Glu 260 265 270 Pro Leu Pro Leu Ser Ala Lys Lys Val Ile Gly Arg Arg Gly Ala Ile 275 280 285 Glu Leu Glu Lys Asp Val Ala Val Asn Leu Gly Val Gly Ala Pro Glu 290 295 300 Tyr Val Ala Ser Val Ala Asp Glu Glu Gly Ile Val Asp Phe Met Thr 305 310 315 320 Leu Thr Ala Glu Ser Gly Ala Ile Gly Gly Val Pro Ala Gly Gly Val 325 330 335 Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr 340 345 350 Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly 355 360 365 Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly 370 375 380 Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr 385 390 395 400 Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val 405 410 415 Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys 420 425 430 Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala 435 440 445 Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe 450 455 460 Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile 465 470 475 480 Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile 485 490 495 Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe 500 505 510 Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser 515 520 <210> 3 <211> 1677 <212> DNA <213> Pseudomonas sp. 6-19 <220> <221> gene <222> (1) .. (1677) <223> PHA synthase <400> 3 atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360 gggcacttcg tgatcaacct catgaccgaa gcgatggcgc cgaccaacac cgcggccaac 420 ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720 cgggtactagctgaca c ggtaccctggcg 720 cgggtactagctgaca c cgacctac atcgaagccc tcaaggaagc ggttgacgtc 840 gttaccgcga tcaccggcag caaagacgtg aacatgctcg gggcctgctc cggcggcatc 900 acttgcactg cgctgctggg ccattacgcg gcgattggcg aaaacaaggt caacgccctg 960 accttgctgg tgagcgtgct tgataccacc ctcgacagcg acgtcgccct gttcgtcaat 1020 gaacagaccc ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080 gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc 1140 aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg gaacaacgac 1200 accacacggt tgcccgcggc gttccacggc gacctgatcg aactgttcaa aaataaccca 1260 ctgattcgcc cgaatgcact ggaagtgtgc ggcaccccca tcgacctcaa gcaggtgacg 1320 gccgacatct tttccctggc cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380 aagtcggcgc aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcag cgggcatatc 1440 cagagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg 1500 gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc atacagattc ctggtggctg 1560 cactggcagg cctggcaggc ccaacgctcg ggcgagctga aaaagtcccc gacaaaactg 1620 ggcagcaagg cgtatccggc aggtgaagcg gcgccaggca cgtacgtgca cgaacgg 1677 <210> 4 <211> 559 <212> PRT <213> Pseudomonas sp. 6-19 <220> <221> PEPTIDE <222> (1)..(559) <223> PHA synthase <400> 4 Met Ser Asn Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn 1 5 10 15 Thr Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu 20 25 30 Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His 35 40 45 Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu 50 55 60 Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Asp Arg Arg Phe Ala 65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Asp Glu Ser Asn 100 105 110 Leu Ala Pro Lys Asp Val Ala Arg Gly His Phe Val Ile Asn Leu Met 115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val 130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val 165 170 175 Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr Glu Gly 180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro 195 200 205 Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu Leu Val Val Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg 245 250 255 Asn Pro Thr Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu 260 265 270 Ala Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys 275 280 285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 290 295 300 Leu Leu Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Asp Val Ala 325 330 335 Leu Phe Val Asn Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp 355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe 405 410 415 Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly Thr 420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser Leu Ala Gly 435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln 450 455 460 Leu Phe Gly Gly Asn Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr 485 490 495 Ser Thr Glu Val Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr 500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln 515 520 525 Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala 530 535 540 Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223 > primer <400> 5 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atgcccggag ccggttcgaa 20 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223 > primer <400> 8 cgttactctt gttactcatg atttgattgt ctctc 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 10 <211 > 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtacgtgcac gaacggtgac gcttgcatga gtg 33 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aacgggaggg aacctgcagg 20 <210> 13 <211> 33 < 212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ctgaccttgc tggtgaccgt gcttgatacc acc 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400 > 14 ggtggtatca agcacggtca ccagcaaggt cag 33 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> < 223> primer <400> 15 cgagcagcgg gcatatcatg agcatcctga acccgc 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcgggttcag gatgctcatg atatgcccgc tgctcg 36 <210> 17 < 211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 atcaacctca tgaccgatgc gatggcgccg acc 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223 > primer <400> 18 ggtcggcgcc atcgcatcgg tcatgaggtt gat 33 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggaattcatg agaaaggttc ccattattac cgcagatga 39 <210> 20 <211 > 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gctctagatt aggacttcat ttccttcaga cccattaagc cttctg 46 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aggcctgcag gcggataaca atttcacaca gg 32 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gcccatatgt ctagattagg acttcatttc c 31 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 cgccggcagg cctgcagg 18 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ggcaggtcag cccatatgtc 20 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 gaattcgtgc tgtcgagccg cgggcatatc 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gatatgcccg cggctcgaca gcacgaattc 30 <210 > 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gggcatatca agagcatcct gaacccgc 28 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gcgggttcag gatgctcttg atatgccc 28 <210> 29 <211> 20 <212> D NA <213> Artificial Sequence <220> <223> primer <400> 29 atgcccggag ccggttcgaa 20 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gaaattgtta tccgcctgca gg 22 <210> 31 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> oligomer <400> 31 atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt aattaaccct 60 cactaaaggg cg 72 <210> 32 <211> 72 <212 > DNA <213> Artificial Sequence <220> <223> oligomer <400> 32 atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt aattaaccct 60 cactaaaggg cg 72 <210> 33 <211> 328 <212> DNA <213> Artificial Sequence <220> <223 > J23 promoter <400> 33 tctagattta cagctagctc agtcctaggt attatgctag cggatcctgt taaaggagca 60 tctgacccat gggcagcagc catcaccatc atcaccacag ccagaattcg agctcggcgc 120 gcctgcaggt cgacaagctt gcggccgcat aatgcttaag tcgaacagaa agtaatcgta 180 ttgtacacgg ccgcataatc gaaatctgac agctagctca gtcctaggta taatgctagc 240 ggtacctgtt aaaggagcat ctgaccatat ggcagatctc aattggatat cggccggcca 300 cgcgatcgct gacgtcttcg aactcgag 328 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tactgaaccg ctctagattt acagctagc 29 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ctttaccaga ctcgagttcg aagacgtca 29 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 tctgacccat ggatgtcttc catgacaaca 30 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 tgtcgacctg caggttactc cacttcctgc ca 32 <210> 38 <211> 1716 <212> DNA <213> Artificial Sequence <220> <223> dld gene from E.coli K-12 MG1655 <400> 38 atgtcttcca tgacaacaac tgataataaa gcctttttga atgaacttgc tcgtctggtg 60 ggttcttcac acctgctcac cgatcccgcttc ggct ggct ggct ggct gctt cggct ggct ggct ggct gt c ggct 120 ggct acct ggct gtgctgaaag cctgcgtcac cgccgacaaa attattctga tgcaggccgc caatacaggc 240 ctgaccgaag gatcgacgcc aaacggtaac gattatgatc gcgatgtcgt tatcatcagc 300 accctgcgtc tcgacaagct gcacgttctt ggcaagggcg aacaggtgct ggcctatccg 360 ggcaccacgc tctattcgct ggaaaaagcc ctcaaaccgc tgggacgcga accgcactca 420 gtgattggat catcgtgtat aggcgcatcg gtcatcggcg gtatttgtaa caactccggc 480 ggctcgctgg tgcaacgtgg cccggcgtat accgaaatgt cgttattcgc gcgtataaat 540 gaagacggca aactgacgct ggtgaaccat ctggggattg atctgggcga aacgccggag 600 cagatcctta gcaagctgga tgatgatcgc atcaaagatg acgatgtgcg tcacgatggt 660 cgtcacgccc acgattatga ctatgtccac cgcgttcgtg atattgaagc cgacacgccc 720 gcacgttata acgccgatcc tgatcggtta tttgaatctt ctggttgcgc cgggaagctg 780 gcggtctttg cagtacgtct tgataccttc gaagcggaaa aaaatcagca ggtgttttat 840 atcggcacca accagccgga agtgctgacc gaaatccgcc gtcatattct ggctaacttc 900 gaaaatctgc cggttgccgg ggaatatatg caccgggata tctacgatat tgcggaaaaa 960 tacggcaaag acaccttcct gatgattgat aagttaggca ccgacaagat gccgttcttc 1020 tttaatctca agggacgc ac cgatgcgatg ctggagaaag tgaaattctt ccgtccgcat 1080 tttactgacc gtgcgatgca aaaattcggt cacctgttcc ccagccattt accgccgcgc 1140 atgaaaaact ggcgcgataa atacgagcat catctgctgt taaaaatggc gggcgatggc 1200 gtgggcgaag ccaaatcgtg gctggtggat tatttcaaac aggccgaagg cgatttcttt 1260 gtctgtacgc cggaggaagg cagcaaagcg tttttacacc gtttcgccgc tgcgggcgca 1320 gcaattcgtt atcaggcggt gcattccgat gaagtcgaag acattctggc gttggatatc 1380 gctctgcggc gtaacgacac cgagtggtat gagcatttac cgccggagat cgacagccag 1440 ctggtgcaca agctctatta cggccatttt atgtgctatg tcttccatca ggattacata 1500 gtgaaaaaag gcgtggatgt gcatgcgtta aaagaacaga tgctggaact gctacagcag 1560 cgcggcgcgc agtaccctgc cgagcataac gtcggtcatt tgtataaagc accggagacg 1620 ttgcagaagt tctatcgcga gaacgatccg accaacagca tgaatccggg gatcggtaaa 1680accagtaaac ggaaaaactg gcaggaagtg gagtaa 1716

Claims (15)

Transformed with a recombinant vector comprising a gene (dld) encoding D-lactate dehydrogenase,
comprising a propionyl-CoA transferase encoding gene and a polyhydroxyalkanoate synthetase encoding gene,
Recombinant microorganisms. The recombinant microorganism according to claim 1, wherein the lactate dehydrogenase A coding gene (ldhA) is inactivated. The recombinant microorganism of claim 1, wherein the recombinant microorganism is Escherichia coli. The recombinant microorganism of claim 1, wherein the gene encoding the D-lactate dehydrogenase is derived from E. coli. The recombinant microorganism of claim 1, wherein the propionyl-CoA transferase encoding gene is derived from Clostridium propionicum . The recombinant microorganism of claim 1, wherein the polyhydroxyalkanoate synthetase encoding gene is phaC derived from a Pseudomonas sp. strain. delete delete delete The recombinant microorganism according to any one of claims 1 to 6, wherein the recombinant microorganism produces a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units. The recombinant microorganism of claim 10, wherein the copolymer including 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units has a lactate monomer content of 6 mol% or less. A method for producing a copolymer comprising 2-hydroxybutyrate and 3-hydroxybutyrate as a repeating unit, comprising the step of culturing the recombinant microorganism of any one of claims 1 to 6. The method according to claim 12, wherein the culturing is performed in a medium supplied with 2-hydroxybutyrate or a precursor thereof, and 3-hydroxybutyrate or a precursor thereof. A copolymer prepared by the method of claim 12 and having a lactate monomer content of 6 mol% or less, comprising 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units. The copolymer according to claim 14, wherein the lactate monomer content is 0 mol%, and the copolymer comprises 2-hydroxybutyrate and 3-hydroxybutyrate as repeating units.

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