KR102002096B1 - Copolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating unit and method for preparing the same - Google Patents

Abstract

Tripolymers comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units and methods for their preparation, and microorganisms for producing the terpolymers and methods for their preparation are provided.

Description

Copolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating unit and method for preparing the same}

Tripolymers comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units and methods for their preparation, and microorganisms for producing the terpolymers and methods for their preparation are provided.

Polyhydroxyalkanoate (PHA) accumulates inside microorganisms for the storage of energy and reducing capacity when microorganisms are rich in carbon sources in the absence of elements necessary for growth such as nitrogen, oxygen, phosphorus and magnesium. It is a natural polyester material. Since PHA has similar properties to synthetic polymers derived from petroleum and shows biodegradability and biocompatibility, PHA has been recognized as a material to replace conventional synthetic plastics.

There are about 150 known PHA monomers, most of which are 3-, 4-, 5- or 6-hydroxyalkanoate (HA), and representative PHA monomers being actively studied 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), 3-hydroxypropionate (3HP), and a medium chain of 6-12 carbon atoms And monomers having a hydroxyl group at carbon positions 3 and 4, such as MCL 3-hydroxyalkanoate of medium chain length (MCL).

An enzyme that plays a key role in synthesizing PHA in microorganisms is PHA synthase, which synthesizes polyesters containing the monomers based on various hydroxyacyl-CoAs. In addition, since 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, metabolic pathways for synthesizing and providing various hydroxyacyl-CoAs that can be used as substrates of PHA synthase and polymer synthesis metabolic pathways using the substrate and PHA synthase are required.

On the other hand, monomers such as lactate or 2-hydroxybutyrate (2HB) having a hydroxyl group at the carbon position 2 are not suitable for the substrate specificity of the PHA synthetase and are naturally or recombinantly. There have been few examples of preparing PHAs and copolymers thereof by the method. Furthermore, no terpolymer PHA polymers produced using lactate, 2HB and 4HB as monomers have yet been reported.

Domestic Patent Registration No. 10-0957777, May 6, 2010

Accordingly, the present invention provides a tripolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units and a technique related to the preparation thereof.

One example provides a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeat units.

Another example is converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA Gene encoding an enzyme to convert to and a polyhydroxyalkanoate synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates It provides a method for producing a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as a repeating unit, comprising culturing a microorganism.

Another example is converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA Genes encoding enzymes that convert to and genes encoding PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates, 4-hydroxy Provided is a microorganism that produces a terpolymer comprising oxybutyrate, 2-hydroxybutyrate and lactate in repeat units.

Another example is converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA Introducing into the cell a gene encoding an enzyme to convert to and a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates. It provides a method for producing a microorganism comprising a tripolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as a repeating unit.

In one aspect, the present invention relates to a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units and a method for preparing the same.

One specific example provides a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units. For example, the present invention relates to 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer.

Another example is converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA A gene encoding a converting enzyme and a gene encoding a polyhydroxyalkanoate synthetase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates It relates to a method for producing a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units, comprising culturing cells.

In another aspect, the present invention relates to a microorganism for producing a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as a repeating unit and a method for producing the same.

Specific examples include converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-. A gene encoding an enzyme converting to CoA, and a gene encoding a PHA synthetase using lactyl-CoA, 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl-CoA as substrates, 4- It relates to a microorganism that produces a terpolymer comprising hydroxybutyrate, 2-hydroxybutyrate and lactate in repeat units.

Another example is converting lactate to lactyl-CoA, converting 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA Introducing into the cell a gene encoding an enzyme to convert to and a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates. It relates to a method for producing a microorganism comprising the 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated in detail.

The term "tripolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating unit" is a monomer wherein 4-hydroxybutyrate, 2-hydroxybutyrate and lactate are polymerized with ester bonds. Refers to a linear polyester containing units. At this time, the polymerization order of each monomer is not particularly limited and may be repeated randomly. For example, 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer, 2-hydroxybutyrate-lactate-4-hydroxybutyrate terpolymer, lactate-4-hydroxybutyrate-2-hydroxy Hydroxybutyrate terpolymer, 4-hydroxybutyrate-lactate-2-hydroxybutyrate terpolymer, 2-hydroxybutyrate-4-hydroxybutyrate-lactate terpolymer, or lactate-2-hydroxybutyrate-4 -Hydroxybutyrate-lactate terpolymer etc. can be illustrated.

The term “convert lactate to lactyl-CoA, convert 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA Enzyme to Convert "is obtained by removing CoA from the CoA donor and transferring it to lactate, 2-hydroxyalkanoate and 4-hydroxyalkanoate, respectively, to lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxy. Refers to an enzyme capable of producing oxyalkanoyl-CoA. Examples of the CoA donor may include acetyl-CoA or acyl-CoA (eg, propionyl-CoA, etc.).

In one embodiment, the enzyme may be propionyl-CoA transferase. In addition, the gene of the enzyme may be derived from Clostridium propionicum (Clostridium propionicum).

For example, convert lactate to lactyl-CoA, convert 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA The gene of the enzyme to switch to

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

(b) a nucleotide sequence of which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;

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

(d) a nucleotide sequence in which Gly335Asp is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;

(e) a nucleotide sequence in which Ala243Thr is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;

(f) a nucleotide sequence in which Asp65Gly is mutated in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;

(g) a nucleotide sequence in which Asp257Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;

(h) a nucleotide sequence in which Asp65Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;

(i) a nucleotide sequence in which Thr199Ile is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and a T669C, A1125G and T1158C mutated in the nucleotide sequence of SEQ ID NO: 1; And

(j) T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala is mutated in the amino acid sequence corresponding to SEQ ID NO: 1

It may have a base sequence selected from the group consisting of.

The term "PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrate" refers to lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4- An enzyme capable of synthesizing a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units using hydroxyalkanoyl-CoA as a substrate.

For example, the enzyme may be PHA synthase (phaC) derived from pseudomonas sp. 6-19.

For example, the PHA synthase is

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

At least one variation 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 It may be composed of a base sequence corresponding to the amino acid sequence.

In another embodiment, the PHA synthase 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 may be composed of a base sequence corresponding to the amino acid sequence comprising a mutation selected from the group consisting of.

The enzymes may include additional variations within the scope that do not alter the activity of the molecule as a whole. For example, amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is 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, Asp / Gly, but is not limited thereto. 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 whose structural stability to heat, pH, etc. of the protein is increased or protein activity is increased by variation or modification on the amino acid sequence.

In addition, the gene encoding the enzyme may include a nucleic acid molecule comprising a codon functionally equivalent 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 such as chemical synthesis or recombinant methods, or may be commercially available.

The microorganism provided herein converts lactate to lactyl-CoA, converts 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkane. A gene encoding an enzyme for converting to noyl-CoA, and a gene encoding a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl-CoA as substrates, The genes may be introduced into a cell by a gene recombinant method.

For example, the microorganism converts lactate to lactyl-CoA, converts 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxy. Genes encoding enzymes that convert alkanoyl-CoA and genes encoding PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates are recombinant vectors. Or genetically engineered to insert the gene on the chromosome.

In addition, the microorganism converts lactate to lactyl-CoA, 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl. Already one of the genes encoding the enzyme converting to -CoA and the gene encoding the PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates The other one may be transformed with a recombinant vector or genetically engineered to insert the gene on a chromosome.

For example, the microorganism may include lactate in a cell containing a gene encoding a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl-CoA as a substrate. Encoding an enzyme that converts lactyl-CoA, converts 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA It may be obtained by transforming a gene.

In another embodiment, the microorganism converts lactate to lactyl-CoA, converts 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and converts 4-hydroxyalkanoate to 4-hydroxyalkane. Genes encoding PHA synthetase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates in cells containing genes encoding enzymes that convert to noyl-CoA It may be obtained by transformation.

Genetic production of microorganisms producing the 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer or by using the microorganisms 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer The production process may include the following steps.

First, convert lactate to lactyl-CoA, convert 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, convert 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA At least one of a gene encoding an enzyme and a gene encoding a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates, Preparing a recombinant vector. The above two genes may be inserted into separate vectors, or may be inserted into one vector.

The term “vector” refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert encoding a target protein in a cell of an individual, wherein the nucleic acid sequence encoding the target protein is introduced into a host cell. It is a means for. As the vector, various types of vectors such as plasmids, viral vectors, bacteriophage vectors, cosmid vectors, and YAC (Yeast Artificial Chromosome) vectors may be used. Recombinant vectors include cloning vectors and expression vectors. Cloning vectors include the origin of replication, eg, the origin of replication of a plasmid, phage or cosmid, and are replicons to which other DNA fragments are attached and to which the attached fragments can be replicated. Expression vectors have been developed for use in synthesizing proteins.

Herein, the vector is not particularly limited as long as it functions to express and produce a desired enzyme gene in various host cells such as prokaryotic or eukaryotic cells, but the gene inserted into the vector and delivered is irreversibly fused into the genome of the host cell. Vectors are preferred that allow long-term stable expression of genes in cells.

Such vectors include transcriptional and translational expression control sequences that allow the gene of interest to be expressed in a selected host. Expression control sequences may include promoters for performing transcription, any operator sequence for controlling such transcription, sequences encoding suitable mRNA ribosomal binding sites, and / or sequences that control termination of transcription and translation. . For example, suitable control sequences for prokaryotes may include promoters, optionally operator sequences, and / or ribosomal binding sites. Suitable regulatory sequences for eukaryotic cells may include promoters, terminators and / or polyadenylation signals. Initiation and termination codons are generally considered to be part of the nucleic acid sequence encoding the protein of interest, and should be functional in the subject and be in frame with the coding sequence when the gene construct is administered. The promoter of the vector may be constitutive or inducible. In addition, if the expression vector is replicable it may include the origin of replication. In addition, enhancers, non-translated regions of the 5 'and 3' ends of the gene of interest, selection markers (e.g., antibiotic resistance markers), or replicable units may be appropriately included. Vectors can self replicate or integrate into host genomic DNA.

Examples of useful expression control sequences include early and late promoters of adenoviruses, monkey virus 40 (SV40), mouse breast tumor virus (MMTV) promoter, long terminal repeat (LTR) promoter of HIV, moroninivirus, cytomegalo Virus (CMV) promoter, Epstein virus (EBV) promoter, Loews sacoma 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, major operator and promoter region of phage lambda, regulatory region of fd code protein, promoter for phosphoglycerate kinase (PGK) or other glycolyase, promoter of phosphatase Such as, for example, Pho5, promoters of the yeast alpha-breeding system and prokaryotic cells or It may include a cell or a nucleus-known configuration and induce other sequences and these various combinations of the genes that control the expression of these virus.

In order to increase the expression level of the transgene in the cell, the desired gene and the transcriptional and translational expression control sequences must be linked to each other so that they can be operated. In general, “operably linked” means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. For example, if the DNA for the pre-sequence or secretion leader is expressed as a shear protein that participates in the secretion of the protein, it may be operably linked to the DNA for the polypeptide, and the promoter or enhancer may be The ribosome binding site may be operably linked to the coding sequence when affecting the transcription of the sequence, or the ribosomal binding site may be operably linked to the coding sequence when affecting the transcription of the sequence, or the ribosomal binding site may facilitate translation. May be operably linked to a coding sequence when arranged to do so. Linking of these sequences can be accomplished by ligation at convenient restriction enzyme sites, and if such sites do not exist, use synthetic oligonucleotide adapters or linkers according to conventional methods. Can be performed.

Those skilled in the art will consider various vectors suitable for the present invention, expression control in consideration of the nature 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, such as antibiotic markers. Sequence, host, etc. can be selected.

Next, transforming the microbial cells using the recombinant vector.

The term “transformation” means that DNA is introduced into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.

The microorganism which can be transformed with the recombinant vector according to the present invention includes both prokaryotic and eukaryotic cells, and a host having high DNA introduction efficiency and high expression efficiency of the introduced DNA can be used. Specific examples, E. coli (e.g., E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli B and E. coli Genus Escherichia, Pseudomonas, Bacillus, Streptomyces, Urbania, Serratia, Providencia, Corynebacterium, Leptospira, Salmonella, Brevi Known eukaryotic and prokaryotic hosts such as bacterial genus, hypomonas genus, chromobacterium genus, nocadia genus, fungi or yeasts and the like can be exemplified, but are not limited thereto. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can 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.

As transformation methods, suitable standard techniques as known in the art, such as 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) and the like, but are not limited thereto. At this time, the circular vector may be cut with an appropriate restriction enzyme and introduced into a linear vector form.

Next, the step of culturing the transformed microorganism to produce a 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer.

By transforming the transformant expressing the recombinant vector in a medium, 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer can be produced and separated in large quantities. The medium and culture conditions may be appropriately selected depending on the type of transformed cells. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and mass production of the terpolymer during the culture. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch cultures.

In one embodiment, the culturing may be performed in a medium containing lactate or a precursor thereof, 2-hydroxybutyrate and / or 4-hydroxybutyrate. Examples of lactate precursors include glucose. In addition, as long as the microorganism capable of biosynthesis of lactate, 2-hydroxybutyrate and 4-hydroxybutyrate from a carbon source such as glucose, the lactate, 2-hydroxybutyrate and / or 4-hydroxybutyrate is not added separately Copolymers can be prepared.

In addition, the medium used for culturing must adequately meet the requirements of the particular strain. The medium may include various carbon sources, nitrogen sources, personnel and trace element components. Carbon sources in the medium include glucose 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, Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, but are not limited thereto. These materials can be used individually or as a mixture. Nitrogen sources in the medium may include peptone, yeast extract, gravy, 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. It is not limited to this. Nitrogen sources can also be used individually or as a mixture. The person in the medium may include, but is not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, the culture medium may include metal salts such as magnesium sulfate or iron sulfate required for growth, or may include 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 in a manner appropriate to the culture during the culturing process.

In addition, if necessary, the pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner. In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Oxygen or an oxygen-containing gas (eg, air) can be injected into the culture to maintain aerobic conditions, and the temperature of the culture can usually be 20 ° C. to 45 ° C., preferably 25 ° C. to 40 ° C. Cultivation can continue until the desired amount of terpolymer is obtained.

Next, the step of recovering the 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer produced.

4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymers produced from recombinant microorganisms can be isolated from cells or culture media by methods well known in the art. Examples of recovery methods for 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymers include centrifugation, sonication, filtration, ion exchange chromatography, high performance liquid chromatography (HPLC), gas Although there exist methods, such as gas chromatography (GC), it is not limited to these examples.

The present invention provides a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units, which is a raw material of biodegradable and biocompatible bioplastics, which is used in electronics, automotive, food, and agriculture. And it can be widely used in the medical field and the like.

Figure 1 shows the construction process and cleavage map of the pPs619C1310-CpPCT540 vector.
2 shows a cleavage map of the pPs619C1249.18H-CPPCT540 vector.
Figure 3 shows the results of gas chromatography analysis of 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer produced from recombinant microorganisms.

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

Example  1. 4- Hydroxybutyrate -2- Hydroxybutyrate - Lactate Terpolymer  Preparation of Recombinant Vectors for Production

1-1. pPs619C1310 - CPPCT540  Preparation of Recombinant Vectors

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

First, PHA synthase (phaC1 _{Ps6 -19)} in order to separate the gene, the total DNA extracted of Pseudomonas species MBEL 6-19 (KCTC 11027BP) and, based on the phaC1 _{Ps6 -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) PCR was performed using the extracted total DNA as a template. The resulting PCR products by electrophoresis, it was confirmed that a gene fragment of 1.7 kb size, corresponding to the phaC1 _{Ps6 -19} gene, and to obtain a phaC1 _{Ps6 -19} gene.

phaC1 _{Ps6 -19} to express the synthase, pSYL105 vector (Lee et al, Biotech Bioeng, 1994, 44:... 1337-1347) Ralstonia in eutropha DNA fragments containing HB-derived PHB-producing operons were cut with BamHI / EcoRI and inserted into the BamHI / EcoRI recognition site of pBluescript II (Stratagene Co., USA) to prepare a pReCAB recombinant vector. The pReCAB vector contains PHA synthase (phaC _RE ) and monomer feeder (phaA _RE). And phaB _RE ) are constantly expressed by the PHB operon promoter. In order to make a phaC1 _{Ps6 -19} synthase gene fragment containing only one BstBI / SbfI recognition site at each end, the BstBI position, which is inherent in the BstBI / SbfI recognition site, was first removed without conversion of amino acids by the site directed mutagenesis (SDM) method. 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'- 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 ACTG CTT GCA TGA GTG 3 ′ (SEQ ID NO: 11), 5′-aac ggg agg gaa cct gca gg −3 ′ (SEQ ID NO: 12)]. pReCAB vector cleaved with BstBI / SbfI to R. eutropha H16, remove the PHA synthase (phaC _RE) and then by inserting the phaC1 _{Ps6 -19} gene obtained above in the BstBI / SbfI recognition site to prepare a recombinant vector pPs619C1-ReAB.

Three amino acid positions affecting SCL (short chain length) activity were found by amino acid sequence sequencing 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)], pPs619C1300- containing phaC1 _{Ps6 -19} synthase variant phaC1 _{Ps6 -19} 300 containing E130D, S325T, Q481M ReAB was prepared.

Propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum was used to construct a system of constant expression of the operon type in which propionyl-CoA transferase was expressed. Used. CP-PCT was used to clone the chromosomal DNA of Clostridium propionicum 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)] was used to fragment obtained by PCR. At this time, NdeI site originally present in wild-type CP-PCT was removed by SDM method for easy cloning, and primer [5'-agg cct gca ggc gga taa caa ttt cac was added to add 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)]. Ralstonia by cutting pPs619C1300-ReAB vector with SbfI / NdeI eutrophus Monomer supply enzyme derived from H16 (phaA _RE And phaB _RE ), and then the pCs619C1300-CPPCT recombinant vector was prepared by inserting the PCR cloned CP-PCT gene into the SbfI / NdeI recognition site.

Next, pPs619C1300-CPPCT prepared above to introduce a random mutagenesis to the CP-PCT gene as a template, primers [5'-CGCCGGCAGGCCTGCAGG-3 '(SEQ ID NO: 23), 5'-GGCAGGTCAGCCCATATGTC -3 '(SEQ ID NO: 24)] was carried out Error-prone PCR in the condition that Mn ^{2 +} is added and the concentration difference of dNTPs is present. Thereafter, PCR was performed under normal conditions using the primers to amplify the PCR fragment containing the random mutation. pPs619C1300-CPPCT vector was digested with SbfI / NdeI to remove wild-type CP-PCT, and then the ligation mixture into which the amplified mutant PCR fragments were inserted at the SbfI / NdeI recognition site was introduced into E. coli JM109 to ~ 10 ⁵ scale CP-PCT library was prepared. The prepared CP-PCT library was grown in a polymer detection medium (LB agar, glucose 20g / L, 3HB 1g / L, Nile red 0.5μg / ml) for 3 days and then screened to determine whether the polymer was produced. More than 80 candidates were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ° C) for 4 days under conditions in which the polymers were produced, and two individuals were analyzed by FACS (Florescence Activated Cell Sorting) analysis. That is, CP-PCT Variant 512 (including nucleic acid substituted A1200G) and CP-PCT Variant 522 (including nucleic acid substituted T78C, T669C, A1125G, and T1158C) were selected. Based on the primary screened mutants (CP-PCT Variant 512, CP-PCT Variant 522), random mutations were performed by the method of Error-prone PCR to obtain various CP-PCT variants. CP-PCT Variant 540 (including Val193Ala and silent mutants T78C, T669C, A1125G, T1158C) was screened twice to prepare the pPs619C1300-CPPCT540 vector.

Further, the above prepared phaC1 _{Ps6 -19} synthase variant (phaC1 _Ps6-19 300) primers [5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3 '( SEQ ID NO: 25) on the basis of, 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 PHA synthase variant derived from Pseudomonas genus MBEL 6-19 with amino acid sequences with E130D, S477F and Q481K variants using the SDM method using ctt gat atg ccc-3 '(SEQ ID NO: 28)] (phaC1 _{Ps6 -19} 310) PPs619C1310-CPPCT540 vector containing was prepared (FIG. 1).

1-2. pPs619C1249 .18H- CPPCT540  Preparation of Recombinant Vectors

Error-prone PCR using primers [5'-ATGCCCGGAGCCGGTTCGAA-3 '(SEQ ID NO: 29) and 5'-GAAATTGTTATCCGCCTGCAGG-3' (SEQ ID NO: 30) using the pPs619C1310-CPPCT540 vector prepared in 1-1 as a template. Was performed. After performing the error-prone PCR, PCR was again performed using the primers to amplify the PCR fragment containing the mutation, and the amplified mutations were inserted into the BstBI / SbfI position of the pPs619C1310-CPPCT540 vector to prepare a library for the variants. It was. The prepared variant library was transformed into E. coli XL-1Blue, and cultured in PHB detection medium (LB agar, glucose 20g / L, Nile red 0.5μg / ml) for 3 days. The final screened variants through incubation and screening were pPs619C1249.18H with amino acid sequences with L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K and A527S. Thus, the recombinant vector pPs619C1249.18H-CPPCT540 vector was prepared (FIG. 2).

Example  2. 4- Hydroxybutyrate -2- Hydroxybutyrate - Lactate Terpolymer  Produce

Recombinant E. coli XL1-Blue was prepared by transforming the recombinant vector prepared in Example 1 by electroporation to E. coli XL1-Blue (Stratagene, USA). Flask incubation was performed to prepare the terpolymer using this. First, for recombinant culture, the recombinant E. coli was cultured with 3 mL of LB medium containing 100 mg / L ampicillin (10 g / L of Bacto ^TM Triptone (BD), 5 g / Bacto ^TM yeast extract (BD)). L, NaCl (amresco) 10 g / L] for 12 hours. For the main culture, 1 ml of the whole culture was prepared with 1 g / L of 4-hydroxybutyrate (4-HB), 1 g / L of 2-hydroxybutyrate (2-HB), 100 mg / L of ampicillin, 10 mg / L of 100 ml MR medium additionally containing thiamine (10 g Glucose per 1 L, 6.67 g KH ₂ PO ₄ , ₄ g (NH ₄ ) 2 HP ₄ , 0.8 g MgSO ₄ .7H ₂ O, 0.8 g citric acid, and 5 mL trace metal solution; Here, trace metal solution is 5M HCl 5mL per 1L, FeSO ₄ · 7H ₂ O 10g, CaCl ₂ 2g, ZnSO ₄ · 7H ₂ O 2.2g, MnSO ₄ · 4H ₂ O 0.5g, CuSO ₄ · 5H ₂ O 1g , (NH ₄ ) 6 Mo ₇ O ₂ · 4H ₂ O 0.1 g, and Na ₂ B ₄ O ₂ · 10H ₂ O 0.02 g), and incubated with stirring at 250 rpm for 3 days at 30 ° C.

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 dried at 80 ° C. for 12 hours. After quantifying the removed cells were reacted with methanol under a sulfuric acid catalyst using chloroform as a solvent at 100 ℃. This was added by mixing distilled water equal to half the volume of chloroform at room temperature and allowed to stand until the two layers were separated. The chloroform layer in which the monomers of the methylated polymer were dissolved in two layers was taken and 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.

As shown in Table 2 and FIG. 3, the GC analysis result confirmed that 4-hydroxybutyrate-2-hydroxybutyrate-lactate terpolymer was produced by recombinant E. coli.

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 volumn / Solvent 1μl / chloroform Initial temp./Time 80 ℃ / 5min Final temp./Time 230 ℃ / 5min Ramp of temp. 7.5 ℃ / min

Total PHA Content (wt%) Polymer (mol%) 4HB 2HB LA 70.8 19.7 27.5 52.9

<110> LG CHEM, LTD. <120> Copolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and          lactate as repeating unit and method for preparing the same <130> DPP20147703KR <160> 30 <170> KopatentIn 1.71 <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 ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140 tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttattgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cctaa 1575 <210> 2 <211> 524 <212> PRT <213> Clostridium propionicum <220> <221> PEPTIDE (222) (1) .. (524) <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 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 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 cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag 780 gaacagcgag agtggggcct gtcgacctac 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 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 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 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> DNA <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

Claims (15)

Tripolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units.
Convert lactate to lactyl-CoA, 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, A gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and
Cultivating a microorganism comprising a gene encoding a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates Steps,
The culturing is carried out in a medium comprising lactate or a precursor thereof, 2-hydroxybutyrate and 4-hydroxybutyrate,
A process for producing a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate as repeating units.
The method of claim 2, wherein the microorganism converts lactate to lactyl-CoA, 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4- Genes encoding enzymes that convert to hydroxyalkanoyl-CoA, and genes encoding PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates Production method obtained by transformation.
The method of claim 2, wherein the lactate is converted to lactyl-CoA, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate is 4-hydroxyalkae. The enzyme converting to noyl-CoA is propionyl-CoA transferase.
The method of claim 2, wherein the lactate is converted to lactyl-CoA, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate is 4-hydroxyalkae. The gene encoding the enzyme that converts to nil-CoA,
(a) the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleotide sequence of which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(c) a nucleotide sequence in which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(d) a nucleotide sequence in which Gly335Asp is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(e) a nucleotide sequence in which Ala243Thr is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(f) a nucleotide sequence in which Asp65Gly is mutated in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(g) a nucleotide sequence in which Asp257Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(h) a nucleotide sequence in which Asp65Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(i) a nucleotide sequence in which Thr199Ile is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and a T669C, A1125G and T1158C mutated in the nucleotide sequence of SEQ ID NO: 1; And
(j) T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala is mutated in the amino acid sequence corresponding to SEQ ID NO: 1
Method of consisting of a base sequence selected from the group consisting of.
The method according to claim 2, wherein the polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Pseudomonas sp. 6-19.
The gene encoding the polyhydroxyalkanoate synthase is according to claim 2,
The amino acid sequence of SEQ ID NO: 4; or
At least one variation in the amino acid sequence of SEQ ID NO: 4 selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R, and A527S Method for producing a base sequence corresponding to the amino acid sequence.
The gene encoding the polyhydroxyalkanoate synthase is according to claim 2,
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) a method of preparation comprising a base sequence corresponding to an amino acid sequence comprising a mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K, and A527S.
delete Enzyme to convert lactate to lactyl-CoA, 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA A gene encoding a gene and a gene encoding a PHA synthase using lactyl-CoA, 2-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates were introduced.
A microorganism which produces a terpolymer comprising 4-hydroxybutyrate, 2-hydroxybutyrate and lactate in repeat units,
Wherein the microorganism is cultured in a medium comprising lactate or a precursor thereof, 2-hydroxybutyrate and 4-hydroxybutyrate.
The method of claim 10, wherein the lactate is converted to lactyl-CoA, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate is 4-hydroxyalkane. The enzyme converting into noyl-CoA is a propionyl-CoA transferase.
The method of claim 10, wherein the lactate is converted to lactyl-CoA, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate is 4-hydroxyalkane. The gene encoding the enzyme that converts to nil-CoA,
(a) the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleotide sequence of which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(c) a nucleotide sequence in which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(d) a nucleotide sequence in which Gly335Asp is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(e) a nucleotide sequence in which Ala243Thr is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(f) a nucleotide sequence in which Asp65Gly is mutated in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(g) a nucleotide sequence in which Asp257Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in a nucleotide sequence of SEQ ID NO: 1;
(h) a nucleotide sequence in which Asp65Asn is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(i) a nucleotide sequence in which Thr199Ile is mutated in an amino acid sequence corresponding to SEQ ID NO: 1 and a T669C, A1125G and T1158C mutated in the nucleotide sequence of SEQ ID NO: 1; And
(j) T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala is mutated in the amino acid sequence corresponding to SEQ ID NO: 1
Microorganisms consisting of a base sequence selected from the group consisting of.
The microorganism according to claim 10, wherein the polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Pseudomonas sp. 6-19.
The gene encoding the polyhydroxyalkanoate synthase is according to claim 10,
The amino acid sequence of SEQ ID NO: 4; or
At least one variation in the amino acid sequence of SEQ ID NO: 4 selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R, and A527S A microorganism consisting of a nucleotide sequence corresponding to the amino acid sequence.
The gene encoding the polyhydroxyalkanoate synthase is according to claim 10,
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) a microorganism, consisting of a base sequence corresponding to an amino acid sequence comprising a mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K, and A527S.

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