KR102623585B1 - Recombinant microorganism and method for preparing terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate and lactate - Google Patents

Abstract

Recombinant microorganism producing a terpolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, and a terpolymer of 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate using the same A manufacturing method is provided.

Description

Recombinant strain and method for preparing terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate and lactate {Recombinant microorganism and method for preparing terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate and lactate}

Recombinant microorganism producing a terpolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, and a terpolymer of 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate using the same A manufacturing method is provided.

Polyhydroxyalkanoate (PHA) is a substance that accumulates inside microorganisms to store energy and reducing ability when carbon sources are abundant while microorganisms lack elements necessary for growth such as nitrogen, oxygen, phosphorus, and magnesium. It is a natural polyester material. Since PHA has similar physical properties to conventional synthetic polymers derived from petroleum and is biodegradable and biocompatible, it is recognized as a material that can replace existing synthetic plastics.

There are more than 150 known monomers of PHA, most of which are 3-, 4-, 5-, or 6-hydroxyalkanoates (HA), and representative PHA monomers that are being actively studied are Lactate (3-hydroxybutyrate: LA), 4-hydroxybutyrate (4HB), 3-hydroxypropionate (3HP), and medium chain length of 6 to 12 carbon atoms. Monomers having hydroxyl groups at carbon positions 3 and 4, such as 3-hydroxyalkanoate (chain length: MCL), may be included.

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

On the other hand, in the case of monomers such as 2-hydroxybutyrate (2HB) and 2-hydroxyisovalerate (2HIV), which have a hydroxy group at the 2 carbon position, the substrate specificity of PHA synthetase is affected. The problem is that it doesn't fit. In order to increase the biosynthesis of 2HB and/or 2HIV, which are useful as monomers of various polymers, it is necessary to introduce enzymes that increase the efficiency of conversion from precursors to 2HB and/or 2HIV.

KR 10-1211767 B1

The present invention relates to an enzyme and/or 2-ke that produces 2-hydroxyalkanoate (e.g., 2-hydroxybutyrate, 2-hydroxyisovalerate) from 2-ketoalkanoate during the metabolic process of microorganisms. By constructing a recombinant microorganism overexpressing an enzyme producing 2-ketobutyrate (e.g., 2-ketobutyrate, 2-ketoisovalerate), 2-hydroxyalkanoate and/or 2-ketoalkano, its precursor, Techniques are provided to enhance the biosynthesis of ates and the efficiency of synthesis of polymers containing them, such as terpolymers containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate.

An example is a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate, a gene encoding an enzyme that produces 2-ketoalkanoate, or a recombinant microorganism into which all of these have been introduced. to provide. The 2-ketoalkanoate may be one or more selected from the group consisting of 2-ketobutyrate and 2-ketoisovalerate. The 2-hydroxyalkanoate may be one or more selected from the group consisting of 2-hydroxybutyrate and 2-hydroxyisovalerate.

Another example provides a composition for producing 2-hydroxyalkanoate, comprising the recombinant microorganism. The 2-hydroxyalkanoate may be one or more selected from the group consisting of 2-hydroxybutyrate and 2-hydroxyisovalerate.

Another example provides a composition for preparing a terpolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, including the recombinant microorganism. The composition enhances the production of a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, and/or increases the production of 2-hydroxybutyrate and/or 2-hydroxyisomer in the terpolymer. This may be to increase the valerate content ratio.

Another example provides a method for producing 2-hydroxybutyrate and/or 2-hydroxyisovalerate, comprising culturing the recombinant microorganism.

Another example is a method for producing a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, comprising culturing the recombinant microorganism, or a method for producing a terpolymer comprising 2-hydroxybutyrate, 2-hydroxy A method of increasing the 2-hydroxybutyrate and/or 2-hydroxyisovalerate content in a terpolymer comprising isovalerate and lactate is provided.

Another example provides a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate. The terpolymer may be prepared by the manufacturing method described above. The terpolymer has a 2-hydroxybutyrate content of about 3 mol% or more, about 5 mol% or more, about 7 mol% or more, about 10 mol% or more, about 12 mol% or more, about 15 mol% or more, about 17 mole. % or more, or about 20 mole % or more. Additionally, the terpolymer may have a content of 2-hydroxyisovalerate of about 5 mol% or more, about 7 mol% or more, about 10 mol% or more, or about 12 mol% or more.

Lactate produced during microbial fermentation and 2-ketobutyrate (2KB) produced during amino acid metabolism are converted to 2-hydroxybutyrate (2HB), which is activated by PHA synthase and coenzyme A converting enzyme. It was confirmed that 2-hydroxybutyrate-lactate copolymer [P(2HB-LA)] was biosynthesized.

In addition, 2-ketoisovalerate (2KIV), another 2-ketoalkanoate (2KA) produced in microorganisms, is used as a precursor to produce 2-hydroxyisovalerate (2). -hydroxyisovalerate; 2HIV) and provides a technology to biosynthesize a new, previously unknown P(2HB-2HIV-LA) tripler from it.

For this purpose, 2-ketoalkanoate (e.g., 2-ketobutyrate and/or 2-ketoisovalerate, etc.) is mixed with 2-hydroxyalkanoate (e.g., 2-hydroxybutyrate (2HB) and/or 2 -Selection of enzymes that can be converted to hydroxyisovalerate (2HIV), etc.) is necessary. Additionally, in order to control the monomer content in the tripolymer polymer, it is necessary to select enzymes involved in the production of 2-ketoalkanoate, a precursor of 2-hydroxyalkanoate.

Accordingly, the present invention provides a method of producing 2-hydroxyalkanoate (2-hydroxybutyrate and/or By producing recombinant microorganisms overexpressing an enzyme with high conversion efficiency to 2-hydroxyisovalerate) and/or an enzyme producing 2-ketoalkanoate (2-ketobutyrate and/or 2-ketoisovalerate) , enhances the biosynthesis of 2-hydroxybutyrate and/or 2-hydroxyisovalerate (thereby increasing the content of 2-hydroxybutyrate and/or 2-hydroxyisovalerate in the cells or culture medium of the recombinant microorganism) ), the efficiency of synthesis of a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, and/or 2-hydroxybutyrate and/or 2-hydroxyisovalerate in the terpolymer. Provides technology to increase content.

An example is a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate, a gene encoding an enzyme that produces 2-ketoalkanoate, or a recombinant microorganism into which all of these have been introduced. to provide. The gene encoding the enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate and the gene encoding the enzyme that produces 2-ketoalkanoate are obtained from recombinant microorganisms (host microorganisms) and heterogeneous microorganisms. It may have originated from it.

In one example, the enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate produces 2-hydroxybutyrate from 2-ketobutyrate and/or 2-hydroxyalkanoate from 2-ketoisovalerate. It may be an enzyme that produces hydroxyisovalerate. For example, the enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate is dehydropantoate reductase, such as Lactococcus lactis- derived 2-dihydropantoate reductase (PanE). You can. Additionally, the enzyme that produces 2-ketoalkanoate may be an enzyme that produces 2-ketobutyrate. For example, the enzyme that produces 2-ketoalkanoate is threonine dehydratase, which produces 2-ketobutyrate from threonine, such as from Corynebacterium glutamicum. It may be L-threonine dehydrogenase (IlvA).

The recombinant microorganism may contain an endogenous and/or foreign (homogeneous or heterologous) CoA transferase encoding gene and PHA synthetase encoding gene.

The recombinant microorganism is used for the production of 2-hydroxybutyrate and/or 2-hydroxyisovalerate, or for the production of a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate. can be used for

Another example provides a composition for producing one or more 2-hydroxyalkanoethites selected from the group consisting of 2-hydroxybutyrate and 2-hydroxyisovalerate, comprising the above recombinant microorganism. The recombinant microorganism for producing the 2-hydroxyalkanoate contains a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate and/or an enzyme that produces 2-ketoalkanoate. The gene encoding may have been introduced.

Another example provides a composition for preparing a terpolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, including the recombinant microorganism. The recombinant microorganism for producing the tripolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate encodes an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate. In addition to the gene encoding and/or the gene encoding the enzyme producing 2-ketoalkanoate, it may include (or be introduced into) a CoA transferase encoding gene and a PHA synthetase encoding gene.

The composition enhances the production of a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, and/or increases the production of 2-hydroxybutyrate and/or 2-hydroxyisomer in the terpolymer. This may be by increasing the content ratio of valerate.

Another example provides a method for producing at least one 2-hydroxyalkanoethyte selected from the group consisting of 2-hydroxybutyrate and 2-hydroxyisovalerate, which includes culturing the recombinant microorganism.

Another example is a method for producing a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, comprising culturing the recombinant microorganism, or a method for producing a terpolymer comprising 2-hydroxybutyrate, 2-hydroxy A method for increasing the 2-hydroxybutyrate and/or 1-hydroxyisovalerate content in a terpolymer comprising isovalerate and lactate is provided.

Another example provides a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate. The terpolymer may be prepared by the manufacturing method described above. The terpolymer has a 2-hydroxybutyrate content of about 3 mol% or more, about 5 mol% or more, about 7 mol% or more, about 10 mol% or more, about 12 mol% or more, about 15 mol% or more, about 17 mole. % or more, or about 20 mol% or more (the upper limit is not particularly limited, but may be about 50 mol% or less, about 40 mol% or less, about 35 mol% or less, or about 30 mol% or less, but is not limited thereto) ; hereinafter, the same applies to the 2-hydroxybutyrate content in the trimer). In addition, the terpolymer may have a content of 2-hydroxyisovalerate of about 5 mol% or more, about 7 mol% or more, about 10 mol% or more, or about 12 mol% or more (the upper limit is not particularly limited, but is about It may be, but is not limited to, 50 mol% or less, about 40 mol% or less, about 35 mol% or less, about 30 mol% or less, about 25 mol% or less, or about 20 mol% or less; hereinafter, 2- in the terpolymer The same applies to the hydroxyisovalerate content).

As used herein, the term “trimer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate” or “2-hydroxybutyrate/2-hydroxyisovalerate/lactate” “Tate tripolymer” refers to a linear polyester containing repeating units in which 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate as monomers are polymerized through ester bonds. At this time, there is no particular limitation on the polymerization order of each monomer and may be repeated randomly. For example, 2-hydroxyisovalerate-2-hydroxybutyrate-lactate terpolymer, 2-hydroxybutyrate-lactate-2-hydroxyisovalerate terpolymer, lactate-2-hydroxyiso Valerate-2-hydroxybutyrate terpolymer, 2-hydroxyisovalerate-lactate -2-hydroxybutyrate terpolymer, 2-hydroxybutyrate-2-hydroxyisovalerate-lactate terpolymer, or Examples include, but are not limited to, 2-hydroxybutyrate-2-hydroxyisovalerate-lactate terpolymer.

The 2-hydroxybutyrate/2-hydroxyisovalerate/lactate tripolymer [P(2HB-2HIV-LA)] is a coenzyme that transfers coenzyme-A (CoA) to 2-hydroxybutyrate and lactate monomers. It can be biosynthesized by -A(CoA) transferase and PHA synthetase, which synthesizes polyhydroxyalkanoate (PHA).

The recombinant microorganism provided herein may contain a CoA transferase encoding gene and a PHA synthase encoding gene for producing 2-hydroxybutyrate/2-hydroxyisovalerate/lactate tripolymer. The genes encoding the CoA transferase and PHA synthase are introduced into microbial cells by conventional genetic recombination methods (e.g., using a recombinant vector containing the CoA transferase encoding gene and the PHA synthase encoding gene separately or together). It may be there.

In addition, the recombinant microorganism has the production of 2-hydroxybutyrate and/or 2-hydroxyisovalerate, the production of 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer, and/or the above three. In order to increase the content of 2-hydroxybutyrate and/or 2-hydroxyisovalerate in the polymer, a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate and/or 2-keto The gene encoding the enzyme that produces toalkanoate may have been engineered to be overexpressed. Overexpression of the gene encoding the enzyme that produces 2-hydroxyalkanoate from the 2-ketoalkanoate and/or the gene that encodes the enzyme that produces 2-ketoalkanoate can be used in recombinant microorganisms and heterogeneous microorganisms. Using a recombinant vector containing, respectively or together, a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate and/or a gene encoding an enzyme that produces 2-ketoalkanoate. It can be performed by conventional genetic recombination methods.

In the above recombinant microorganism, a gene encoding a CoA transferase and a gene encoding a PHA synthetase, a gene encoding an enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate, and/or a 2-ketoalkanoate Genes encoding enzymes that produce ate can be included (inserted) in separate recombinant vectors, or two or more of them can be included (inserted) together in one vector and introduced into the microorganism (host microorganism to be recombined). there is.

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) Base sequence of SEQ ID NO: 1;

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

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

(d) Including the mutation of A1200G in the base sequence of SEQ ID NO: 1, and G335A in the amino acid sequence corresponding to SEQ ID NO: 1 (meaning a mutation in which the 335th amino acid Gly is replaced with Ala, in the amino acid sequence mutation expression described below) The same applies) a base sequence encoding an amino acid sequence containing a mutation;

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

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

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

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

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

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

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

The polyhydroxyalkanoate (PHA) synthase is an enzyme that biosynthesizes polyhydroxyalkanoate using CoA and thioester of hydroxy fatty acid as a substrate, and uses fatty acids with 3-5 carbon atoms. It may be a type that uses fatty acids (e.g., from various bacteria such as Cupriavidus necator and Alcaligenes latus) and a type that uses fatty acids with 6 to 14 carbon atoms (e.g., from the genus Pseudomonas).

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

For example, the PHA synthetase,

Amino acid sequence of SEQ ID NO: 4; or

Containing one or more mutations selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R and A527S in the amino acid sequence of SEQ ID NO: 4 amino acid sequence

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

The enzyme that produces 2-hydroxyalkanoate from 2-ketoalkanoate produces 2-hydroxybutyrate from 2-ketobutyrate and/or 2-hydroxyisovalerate from 2-ketoisovalerate. It may be an enzyme that produces valerate, for example, it may be a microorganism (host microorganism to be recombinant) or a dehydropantoate reductase derived from a heterogeneous microorganism. In one example, the dihydropantoate reductase may be 2-dihydropantoate reductase (PanE; EC_1.1.1.169; SEQ ID NO. 39) derived from Lactococcus lactis , and the gene encoding this may have, for example, sequence It may include the nucleic acid sequence numbered 38 (NC_002662.1).

The enzyme that produces 2-ketoalkanoate may be an enzyme that produces 2-ketobutyrate, for example, threonine dehydratase that produces 2-ketobutyrate from threonine. In one example, the threonine dehydrogenase is derived from Corynebacterium glutamicum It may be L-threonine dehydrogenase (IlvA; EC_4.3.1.19; SEQ ID NO: 41), and the gene encoding it may include the nucleic acid sequence of SEQ ID NO: 40 (NC-003450.3).

The enzymes may contain additional mutations as long as they do not alter the overall activity of the molecule. For example, amino acid exchanges in proteins and peptides that do not overall alter the activity of the molecule are known in the art. For example, commonly occurring exchanges include 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, and Asp/Gly, but are not limited thereto. In some cases, the protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. In addition, it may include enzyme proteins with increased structural stability against heat, pH, etc. or increased protein activity due to mutations or modifications in the amino acid sequence.

In addition, the gene encoding the enzyme may include a nucleic acid molecule containing a functionally equivalent codon, a codon encoding the same amino acid (due to codon degeneracy), or a codon encoding a biologically equivalent amino acid. there is. The nucleic acid molecules can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available ones can be used.

“Vector” refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert encoding a target protein in the cells of an individual, and a means for introducing the nucleic acid sequence encoding the target protein into a host cell. This happens. The vector may be one or more types selected from the group consisting of various vectors such as viral vectors such as plasmids, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, bacteriophage vectors, cosmid vectors, and YAC (Yeast Artificial Chromosome) vectors. there is. In one example, the plasmid vector is pBlue (e.g., pBluescript II KS(+)), pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, It may be one or more types selected from the group consisting of pGEX series, pET series, pUC19, etc., and the bacteriophage vector may be one or more types 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 foreign genes of interest. A cloning vector is a replicon that contains an origin of replication, for example, a plasmid, phage, or cosmid, to which another DNA fragment can be attached so that the attached fragment can be replicated. Expression vectors have been developed to be used to synthesize proteins.

In the present specification, the vector is not particularly limited as long as it can express and produce the desired enzyme gene in various host cells such as prokaryotic cells or eukaryotic cells, but the gene inserted and delivered into the vector does not enter the genome of the host cell. It is better to fuse reversibly so that gene expression continues stably for a long period of time within the cell.

These 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 promoters to effect transcription, optional operator sequences to regulate such transcription, sequences encoding suitable mRNA ribosome binding sites, and/or sequences that regulate termination of transcription and translation. . For example, suitable regulatory sequences for prokaryotes may include a promoter, optionally an operator sequence and/or a ribosome binding site. Regulatory sequences suitable for eukaryotic cells may include promoters, terminators and/or polyadenylation signals. Initiation codons and stop codons are generally considered to be part of the nucleic acid sequence encoding the protein of interest, 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. Additionally, if it is a replicable expression vector, it may include an origin of replication. In addition, it may appropriately include an enhancer, an untranslated region at the 5' and 3' ends of the gene of interest, a selection marker (eg, antibiotic resistance marker), or a replicable unit. Vectors can self-replicate or integrate into host genomic DNA.

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

In order to increase the expression level of a transgene in a cell, the gene of interest and the transcriptional and translational expression control sequences must be operably linked to each other. Generally, “operably linked” means that the linked DNA sequences are in contact and, in the case of a secreted leader, in contact and within reading frame. For example, the DNA for a pre-sequence or secretion leader can be operably linked to the DNA for a polypeptide when expressed as a pre-protein that participates in secretion of the protein, and a promoter or enhancer may be operably linked to the DNA for the polypeptide. may be operably linked to a coding sequence if it affects transcription of the sequence, or a ribosome binding site may be operably linked to a coding sequence if it affects transcription of the sequence, or the ribosome binding site may facilitate translation. When arranged to do so, it can be operably linked to a coding sequence. Linking of these sequences can be accomplished by ligation at convenient restriction enzyme sites, or, if such sites do not exist, by using synthetic oligonucleotide adapters or linkers according to conventional methods. It can be performed by doing this.

Those skilled in the art will consider the nature of the host cell, the copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, and control the expression of various vectors suitable for the present invention. 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” refers to introducing a gene of interest into a host microorganism so that the gene can be replicated as an extrachromosomal factor or by completing chromosomal integration.

Microorganisms that can be used as host microorganisms can be selected from the group consisting of prokaryotic cells and eukaryotic cells. Typically, microorganisms that have high DNA introduction efficiency and high expression efficiency of the introduced DNA can be used as host microorganisms. The host microorganism specifically includes Escherichia coli (e.g., E.coli DH5a, E.coli JM101, E.coli K12, E.coli W3110, E.coli X1776, E.coli B, and E.coli XL1-Blue). Includes Escherichia, Pseudomonas, Bacillus, Streptomyces, Erwinia, Serratia, Providencia, Corynebacterium, Leptospira, Salmonella, Brevibacteria, and Hypomonas. Examples include, but are not limited to, well-known eukaryotic and prokaryotic hosts such as genus Chromobacterium, Nocadia, fungi, or yeast. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.

Additionally, for the purpose of the present invention, the host cell may be a microorganism that has a pathway to biosynthesize hydroxyacyl-CoA from a carbon source.

Transformation methods include suitable standard techniques as known in the art, such as electroporation, electroinjection, microinjection, and 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), etc. may be used, but are not limited thereto. At this time, the circular vector can be cut with an appropriate restriction enzyme and introduced in the form of a linear vector.

The transformed recombinant microorganism can be cultured to produce 2-hydroxybutyrate and/or 2-hydroxyisovalerate, or 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer. .

The recombinant microorganism into which the recombinant vector was introduced is cultured in an appropriate medium to produce 2-hydroxybutyrate and/or 2-hydroxyisovalerate, and/or 2-hydroxybutyrate/2-hydroxyisovalerate/lactate. Tripolymers can be produced effectively. The medium and culture conditions used at this time can be appropriately selected and used according to the type of recombinant microorganism. During culture, conditions such as temperature, pH of the medium, and culture time can be appropriately adjusted to suit cell growth and triplex production. Examples of the above culture methods include, but are not limited to, batch, continuous, and fed-batch culture.

In one embodiment, the culture is performed to produce 2-ketoalkanoate (2-ketobutyrate, 2-ketoiso), which is a precursor of 2-hydroxyalkanoate (2-hydroxybutyrate, 2-hydroxyisovalerate). It may be performed in a medium containing glucose and/or threonine, which are precursors of valerate), and/or 2-ketoalkanoate. In addition, if the recombinant microorganism can biosynthesize lactate from a carbon source such as glucose, the terpolymer can be produced by supplying a conventional carbon source without separately adding lactate and/or its precursor.

In addition, the medium used for culture must appropriately meet the requirements of the specific strain. The medium may contain various carbon sources, nitrogen sources, phosphorus and trace element components. Carbon sources in the medium include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil, and palmitic acid, stearic acid, and linoleic acid. Examples may include one or more selected from the group consisting of fatty acids, glycerol, alcohols such as ethanol, and organic acids such as acetic acid, but are not limited thereto. These substances can be used individually or in mixtures. Nitrogen sources in the medium include amino acids, peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. However, it is not limited to this. Nitrogen sources can also be used individually or in mixtures. Persons in the medium include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. Additionally, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth, or may contain essential growth substances such as amino acids and vitamins, but is not limited thereto. The above-mentioned raw materials can be added to the culture in an appropriate manner, batchwise or continuously, during the cultivation process.

Additionally, if necessary, the pH of the culture can be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid in an appropriate manner. Additionally, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain aerobic conditions, and the temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C. Cultivation can be continued until maximum production of the desired polymer is obtained.

The method for producing 2-hydroxybutyrate and/or 2-hydroxyisovalerate, or the method for producing 2-hydroxybutyrate/2-hydroxyisovalerate/lactate tripolymer provided in the present invention involves culturing a recombinant microorganism. After the step, the produced 2-hydroxybutyrate and/or 2-hydroxyisovalerate, or 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer is recovered (or separated) from the culture. Or purification) may additionally be included.

2-Hydroxybutyrate and/or 2-hydroxyisovalerate, or 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer produced from recombinant microorganisms, by methods well known in the art. It can be isolated from cells or culture medium. Examples of methods for recovering the 2-hydroxybutyrate and/or 2-hydroxyisovalerate, or 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer include centrifugation, ultrasonic disruption, and filtration. , ion exchange chromatography, high performance liquid chromatography (HPLC), gas chromatography (GC), etc., but are not limited to these examples.

The present invention relates to 2-hydroxyalkanoate (2-hydroxybutyrate, 2-hydroxyisovalerate), and/or hemp containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate. Provides a technology for efficiently producing polymers, and by using the technology, intracellular and/or intracellular production of 2-hydroxyalkanoate (2-hydroxybutyrate, 2-hydroxyisovalerate), Production amount of 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer, and/or 2-hydroxyalkanoate (2-hydroxybutyrate, 2-hydroxyisovalerate) in the terpolymer The content can be increased.

Figure 1 shows the production process and cleavage map of the pPs619C1310-CpPCT540 vector.
Figure 2 shows a cleavage map of the pPs619C1249.18H-CPPCT540 vector.
Figure 3 shows the production process and cleavage map of the pCDFJ23- ilvA vector.
Figure 4 shows the construction process and cleavage map of the pCDFJ23- panE vector.
Figure 5 shows the production process and cleavage map of the pCDFJ23- ilvA_panE vector.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of recombinant vector for producing 2-hydroxybutyrate/2-hydroxyisovalerate/lactate tripolymer

1-1. Preparation of pPs619C1310-CPPCT540 recombinant vector

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

First, in order to isolate the PHA synthase (phaC1Ps6-19) gene, the total DNA of Pseudomonas genus MBEL 6-19 (KCTC 11027BP) was extracted, and based on the phaC1Ps6-19 gene sequence (SEQ ID NO: 3), primers [5 '-GAGAGACAATCAAATCATGAGTAACAAGAGTAACG-3' (SEQ ID NO: 5), 5'-CACTCATGCAAGCGTCACCGTTCGTGCACGTAC-3' (SEQ ID NO: 6)] were prepared, and PCR was performed using the extracted total DNA as a template. The obtained PCR product was subjected to electrophoresis to identify a 1.7 kb gene fragment corresponding to the phaC1Ps6-19 gene, and the phaC1Ps6-19 gene was obtained.

To express phaC1Ps6-19 synthetase, a DNA fragment containing the PHB production operon from Ralstonia eutropha H16 was transformed into BamHI/EcoRI in the pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347). The pReCAB recombinant vector was prepared by cutting and inserting into the BamHI/EcoRI recognition site of pBluescript II (Stratagene Co., USA). In the pReCAB vector, PHA synthetase (phaCRE) and monomer supply enzymes (phaARE and phaBRE) are constitutively expressed by the PHB operon promoter. To create a phaC1Ps6-19 synthetase gene fragment containing only one BstBI/SbfI recognition site at each end, the inherent BstBI site was first removed without amino acid conversion using SDM (site directed mutagenesis), and the BstBI/SbfI recognition site was removed. To add primers [5'-ATGCCCGGAGCCGGTTCGAA-3' (SEQ ID NO: 7), 5'-CGTTACTCTTGTTACTCATGATTTGATTGTCTCTC-3' (SEQ ID NO: 8), 5'-GAGAGACAATCAAATCATGAGTAACAAGAGTAACG-3' (SEQ ID NO: 9), 5'-CACTCATGCAAGCGTCACCGTTCGTGCACGTAC- Overlapping PCR was performed using 3' (SEQ ID NO: 10), 5'-GTACGTGCACGAACGGTGACGCTTGCATGAGTG-3' (SEQ ID NO: 11), and 5'-AACGGGAGGGAACCTGCAGG-3' (SEQ ID NO: 12). The pReCAB vector was digested with BstBI/SbfI to remove R.eutropha H16 PHA synthase (phaCRE), and then the phaC1Ps6-19 gene obtained above was inserted into the BstBI/SbfI recognition site to prepare the pPs619C1-ReAB recombinant vector.

Three amino acid positions that affect SCL (short chain length) activity were found through amino acid sequence analysis, and primers [5'-CTGACCTTGCTGGTGACCGTGCTTGATACCACC-3' (SEQ ID NO: 13), 5'-GGTGGTATCAAGCACGGTCACCAGCAAGGTCAG-3' (SEQ ID NO: 14) ), 5'-CGAGCAGCGGGCATATCATGAGCATCCTGAACCCGC-3' (SEQ ID NO: 15), 5'-GCGGGTTCAGGATGCTCATGATATGCCCGCTGCTCG-3' (SEQ ID NO: 16), 5'-ATCAACCTCATGACCGATGCGATGGCGCCGACC-3' (SEQ ID NO: 17), 5'-GGTCGGCGCCATCGCATCGGTCATGAGGTTGAT-3 '(Sequence Using the SDM method using [No. 18)], pPs619C1300-ReAB containing phaC1Ps6-19300, a phaC1Ps6-19 synthetase variant containing E130D, S325T, and Q481M, was prepared.

Here, in order to construct a constitutive expression system in the form of an operon in which propionyl-CoA transferase is co-expressed, propionyl-CoA transferase (CP-PCT) from Clostridium propionicum was used. used. CP-PCT is a fragment obtained by PCR of chromosomal DNA of Clostridium propionicum using primers [5'-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3' (SEQ ID NO. 19), 5'-GCTCTAGATTAGGACTTCATTTCCTTCAGACCCATTAAGCCTTCTG-3' (SEQ ID NO. 20)] used. At this time, the NdeI site originally present in wild-type CP-PCT was removed using the SDM method for ease of cloning, and to add the SbfI/NdeI recognition site, primer [5'-AGGCCTGCAGGCGGATAACAATTTCACACAGG-3' (SEQ ID NO: 21) was used. , 5'-GCCCATATGTCTAGATTAGGACTTCATTTCC-3' (SEQ ID NO: 22)] was used to perform overlapping PCR. The pPs619C1300-ReAB vector was digested with SbfI/NdeI to remove the monomer supply enzymes (phaARE and phaBRE) derived from Ralstonia eutrophus H16, and then the PCR-cloned CP-PCT gene was inserted into the SbfI/NdeI recognition site to create the pPs619C1300-CPPCT recombinant vector. was manufactured.

Next, in order to introduce random mutagenesis into the CP-PCT gene, pPs619C1300-CPPCT prepared above was used as a template, and primers [5'-CGCCGGCAGGCCTGCAGG-3' (SEQ ID NO. 23), 5'-GGCAGGTCAGCCCATATGTC -3' (SEQ ID NO: 24)] was used to perform error-prone PCR under conditions where Mn2+ was added and there was a difference in the concentration of dNTPs. Afterwards, PCR was performed under normal conditions using the above primers to amplify the PCR fragment containing the random mutation. The pPs619C1300-CPPCT vector was cut with SbfI/NdeI to remove wild-type CP-PCT, and then the amplified mutant PCR fragment was inserted into the SbfI/NdeI recognition site to create a ligation mixture and introduced into E. coli JM109 to produce ~105 cells. A CP-PCT library was created. The CP-PCT library produced above was grown for 3 days on polymer detection medium (LB agar, glucose 20g/L, LA 1g/L, Nile red 0.5㎍/ml), and then a screening operation was performed to determine whether polymers were produced. Approximately 80 candidates were initially selected. These candidates were cultured in liquid for 4 days under conditions in which polymers are produced (LB agar, glucose 20g/L, LA 1g/L, ampicillin 100mg/L, 37℃), 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 substitutions T78C, T669C, A1125G, and T1158C) were selected. Based on the first selected mutants (CP-PCT Variant 512, CP-PCT Variant 522), random mutations were performed using the Error-prone PCR method to obtain various CP-PCT variants. CP-PCT Variant 540 (including Val193Ala and silent mutations T78C, T669C, A1125G, and T1158C) was subjected to secondary selection to prepare the pPs619C1300-CPPCT540 vector.

In addition, based on the phaC1Ps6-19 synthase mutant (phaC1Ps6-19300) prepared above, primers [5'-GAATTCGTGCTGTCGAGCCGCGGGCATATC-3' (SEQ ID NO: 25), 5'-GATATGCCCGCGGCTCGACAGCACGAATTC-3' (SEQ ID NO: 26), 5' -GGGCATATCAAGAGCATCCTGAACCCGC-3' (SEQ ID NO: 27), 5'-GCGGGTTCAGGATGCTCTTGATATGCCC-3' (SEQ ID NO: 28)] derived from Pseudomonas genus MBEL 6-19 with amino acid sequences in which E130D, S477F, and Q481K were mutated using the SDM method. The pPs619C1310-CPPCT540 vector containing the PHA synthase mutant (phaC1Ps6-19310) was prepared (Figure 1).

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

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

Example 2. Preparation of recombinant strains for producing 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer

E. coli XL1-blue (Stratagene, USA), a recombinant vector (pCDFJ23- ilvA ; Figure 3) containing the L-threonine dehydratase gene ( ilvA ) (NC_003450.3; SEQ ID NO: 40) derived from Corynebacterium glutamicum , derived from Lactococcus lactis By introducing a recombinant vector (pCDFJ23- panE ; Figure 4) containing the 2-Dehydropantoate reductase gene ( panE ) (NC_002662.1; SEQ ID NO: 38), or a recombinant vector containing both (pCDFJ23- ilvA_panE ; Figure 5) , an E. coli mutant overexpressing ilvA, an E. coli mutant overexpressing panE, and an E. coli mutant overexpressing both panE and ilvA (panE+ilvA) were produced, respectively.

2.1. Construction of ilvA gene overexpression strain

The pCDFJ23 vector (2.3kb) was constructed to express the L-threonine dehydratase enzyme. The pCDFJ23 vector is made by cutting the DNA fragment containing the two T7 promoters from pCDFDuet ^TM -1 (Novagen Co., USA, 3.7kb) with XbaI/XhoI, and constitutively expressing the J23101 and J23108 promoters at the It was manufactured by inserting it. The size of the inserted DNA fragment (promoter) is 328bp (SEQ ID NO: 31). For insertion into the J23101 and J23108 promoters, primers [5'-TACTGAACCGCTCTAGATTTACAGCTAGC-3' (SEQ ID NO: 32) and 5'-CTTTACCAGACTCGAGTTCGAAGACGTCA-3' (SEQ ID NO: 33)] to which XbaI/XhoI recognition sites were added were used.

The L-threonine dehydratase gene ( ilvA ) was inserted into the NcoI/EcoRI recognition site of the constructed CDFJ23 vector. To isolate the L-threonine dehydratase gene (ilvA), the total DNA of Corynebacterium glutamicum was extracted, and based on the ilvA gene sequence (NC_003450.3; SEQ ID NO. 40), primers containing NcoI/EcoRI recognition sites were used [5'-TCTGACCCATGGAATGAGTGAAACATACGTTT-3' (SEQ ID NO: 34), 5'-GAGCTCGAATTCTTATGTCAGGTATTCGTATT-3' (SEQ ID NO: 35)] were prepared, and PCR was performed using the extracted total DNA as a template. The obtained PCR product was subjected to electrophoresis to identify a 1.3 kb gene fragment corresponding to the ilvA gene, and the ilvA gene was obtained. pCDFJ23- ilvA (3.6 kb; see Figure 3) was constructed by inserting the ilvA gene into the pCDFJ23 vector.

The pCDFJ23- ilvA prepared above was introduced into E. coli XL1-blue (Stratagene, USA) by electroporation to produce an E. coli mutant overexpressing ilvA.

2.2. Construction of panE gene overexpression strain

The 2-Dehydropantoate reductase gene ( panE ) (NC_002662.1; SEQ ID NO: 38 ; 0.9 kb) derived from Lactococcus lactis was cloned into the SbfI and HindIII positions of pCDFJ23 prepared in Example 2.1. Primers containing SbfI and HindIII recognition sites [5'-GGCGCGCCTGCAGGACAGGATGTTAAAGGAGCATCTGACATGAGAATTACAATTGCCG-3' (SEQ ID NO. 36), 5'-GGCCGCAAGCTTTTATTTTGCTTTTTAATAACT-3' (SEQ ID NO. 37)] were used. The panE gene was inserted here to create pCDFJ23- panE (3.2 kb; see Figure 4).

The pCDFJ23- panE prepared above was introduced into E. coli XL1-blue (Stratagene, USA) by electroporation to produce an E. coli mutant overexpressing panE .

2.3 Construction of vectors for overexpression of ilvA and panE genes

The panE gene was cloned into the SbfI/HindIII recognition site of pCDFJ23- ilvA (3.6 kb; see Figure 3) constructed in 2.1, thereby constructing pCDFJ23- ilvA_panE (4.5 kb; see Figure 5). At this time, primers containing SbfI and HindIII recognition sites [5'-GGCGCGCCTGCAGGACAGGATGTTAAAGGAGCATCTGACATGAGAATTACAATTGCCG-3' (SEQ ID NO: 36), 5'-GGCCGCAAGCTTTTATTTTGCTTTTTAATAACT-3' (SEQ ID NO: 37)] were used.

The pCDFJ23- ilvA_panE constructed above was introduced into E. coli XL1-blue (Stratagene, USA) by electroporation to produce an E. coli mutant overexpressing ilvA + panE .

2.4. Preparation of recombinant strains for production of 2-hydroxybutyrate/2-hydroxyisovalerate/lactate tripolymer

E. coli XL1-blue (Stratagene, USA), and the ilvA overexpressing E. coli variant prepared in Example 2.1, the panE overexpressing E. coli variant prepared in Example 2.2, and the panE + ilvA overexpressing E. coli variant prepared in Example 2.3. Each was transformed by electroporation using the recombinant vector pPs619C1249.18H-CPPCT540 prepared in Example 1.2, thereby producing a recombinant strain for producing 2HB-2HIV-LA triplex.

Example 3. Preparation of 2-hydroxybutyrate/2-hydroxyisovalerate/lactate terpolymer

The recombinant strain prepared in Example 2.4 was cultured in 3 mL of LB medium containing 100 mg/L ampicillin and 25 mg/L streptomycin [Bacto ^TM Triptone (BD) 10 g/L, Bacto ^TM yeast extract]. (BD) 5g/L, NaCL (amresco) 10g/L] for 12 hours (seed culture).

1 ml of the obtained pre-culture was added to 100 ml MR medium (10 g of Glucose per 1 L of medium, KH ₂ PO ₄ 6.67g, (NH ₄ )2HPO ₄ 4g, MgSO ₄ ·7H ₂ O 0.8g, citric acid 0.8g, and trace metal solution 5mL; Here, trace metal solution is 5M HCl 5mL, FeSO _4· 7H ₂ per 1L. 10 g of O, 2 g of CaCl ₂ , 2.2 g of ZnSO _4· 7H ₂ O, 0.5 g of MnSO ₄ ·4H ₂ O, 1 g of CuSO ₄ ·5H ₂ O, 0.1 g of (NH ₄ )6Mo ₇ O ₂ ·4H ₂ O, and Na. ₂ B ₄ O ₂ ·10H ₂ O (containing 0.02 g) was inoculated and the main culture was performed at 30°C for 4 days while stirring at 250 rpm.

The culture medium was centrifuged at 4000 rpm for 10 minutes at 4°C to recover the cells, washed twice with a sufficient amount of distilled water, and dried at 80°C for 12 hours. After quantifying the removed bacteria, they were reacted with methanol under a sulfuric acid catalyst at 100°C using chloroform as a solvent. This was mixed by adding distilled water in a volume equivalent to half that of chloroform at room temperature and 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 components were analyzed by gas chromatography (GC). Benzoate was used as an internal standard. 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 to flow 1ml/min Column head pressure 29kPa Injection port mode Splitless Injection volume/Solvent 1㎕/chloroform Initial temp./Time 80℃/5min Final temp./Time 230℃/5min Ramp of temp. 7.5℃/min

The results obtained from the GC analysis are shown in Table 2 below:

Strain (with 619C1_249 + CPPCT_540) LA mol % 2HB mol% 2HIV mol% P(LA-2HB-2HIV) content (wt%) Control 98±1.4 2.0±0.6 - 5.2±2.1 IlvA 86.8±1.7 13.2±1.3 - 6.0±2.3 PanE 86.8±1.8 4.0±0.3 9.1±1.7 26.1±2.8 IlvA + PanE 64.4±1.8 22.6±1.2 12.9±0.5 22.5±2.1

(Control: Recombinant E. coli in which pPs619C1249.18H-CPPCT540 was introduced into E. coli XL1-blue (Stratagene, USA); '-': not detected (meaning not present (0 mole), small or very small amount present))

As shown in Table 2, when overexpressing PanE, the polymer production of recombinant E. coli increases about 5-fold compared to the control group, and the sum of the molar contents of 2HB and 2HIV in the trimer is about 13% (2HB: 4 mol%) , 2HIV: 9 mol%), an increase of about 6 times compared to the control group (2 mol%). In recombinant E. coli that overexpressed IlvA, the molar content of 2HB in the polymer increased about 6-fold compared to the control group. In addition, in recombinant E. coli that overexpressed IlvA and PanE together, compared to the control group, tripler production increased about 4-fold, the molar content of 2HB in the tripler increased about 11-fold, and the sum of the molar contents of 2HB and 2HIV was It increased about 17 times.

Claims (16)

Transformed with a recombinant vector containing both the dihydropantoate reductase encoding gene and the threonine dehydrogenase encoding gene,
Contains a gene encoding propionyl-CoA transferase and a gene encoding polyhydroxyalkanoate synthase,
The propionyl-CoA transferase encoding gene encodes a base sequence containing T78C, T669C, A1125G and T1158C mutations in the base sequence of SEQ ID NO: 1 and an amino acid sequence containing the Val193Ala mutation in the amino acid sequence corresponding to SEQ ID NO: 1. Contains a base sequence,
The polyhydroxyalkanoate synthetase encoding gene has a base sequence encoding an amino acid sequence including the mutations L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K and A527S in the amino acid sequence of SEQ ID NO: 4. With recombinant microorganisms, including,
The recombinant microorganism produces a terpolymer containing 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate. The recombinant microorganism according to claim 1, wherein the dihydropantoate reductase encoding gene is the 2-dihydropantoate reductase gene (panE) derived from Lactococcus lactis . The method of claim 1, wherein the gene encoding threonine dehydrogenase is derived from Corynebacterium glutamicum. A recombinant microorganism with an L-threonine dehydrogenase gene (ilvA). The recombinant microorganism according to claim 1, wherein the recombinant microorganism is Escherichia coli. The recombinant microorganism according to claim 1, wherein the propionyl-CoA transferase encoding gene is derived from Clostridium propionicum . The recombinant microorganism according to claim 1, wherein the polyhydroxyalkanoate synthase encoding gene is phaC derived from a strain of the genus Pseudomonas. delete delete delete delete The method according to any one of claims 1 to 6, wherein the terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate has a 2-hydroxybutyrate content of 3 mol% or more. Recombinant microorganism, which is less than mol%. A method for producing a terpolymer comprising 2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate, comprising culturing the recombinant microorganism of any one of claims 1 to 6. delete delete delete delete