KR20170028186A - Copolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating unit and method for preparing the same - Google Patents

Abstract

Hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, a process for their preparation, and microorganisms for the production of the terpolymers and processes for their preparation are provided.

Description

[0001] The present invention relates to a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, and a process for preparing the same. the same}

Hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, a process for their preparation, and microorganisms for the production of the terpolymers and processes for their preparation are provided.

Polyhydroxyalkanoate (PHA) is a microorganism that accumulates in microorganisms for the storage of energy and reducing ability when a microorganism is abundant in a carbon source in a state in which an element necessary for growth of nitrogen, oxygen, phosphorus, It is a natural polyester material. PHA has been recognized as a substitute for conventional synthetic plastics because it exhibits similar biodegradability and biocompatibility with synthetic polymer derived from petroleum.

Most of the known monomers are PHA monomers such as 3-, 4-, 5- or 6-hydroxyalkanoate (HA), and PHA monomers which are actively studied 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), 3-hydroxypropionate (3HP), and an intermediate chain having 6 to 12 carbon atoms Monomers having a hydroxyl group at carbon positions 3 and 4, such as 3-hydroxyalkanoate of medium chain length (MCL), and the like.

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

On the other hand, monomers such as lactate or 2-hydroxybutyrate (2HB) having a hydroxy group at the 2-carbon position are not suitable for the substrate specificity of the PHA synthase, and naturally or recombinantly There were few examples of the preparation of PHA and its copolymer. Furthermore, there has not been reported a terpolymer PHA polymer produced using 2HB, 3HB and 4HB as monomers.

Domestic patent registration 10-0957777, May 6, 2010

Accordingly, the present invention provides a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, and a technique for preparing the same.

An example provides a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as recurring units.

Another example is that the activity of lactate dehydrogenase is weakened or deficient, the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 2- Hydroxyalkanoyl-CoA, converting 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA, converting 4-hydroxyalkanoate into 4-hydroxyalkanoyl-CoA Encoding a polyhydroxyalkanoate synthase using 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, comprising the step of culturing a microorganism containing the gene.

Another example is that the activity of lactate dehydrogenase is weakened or deficient, the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 2- Hydroxyalkanoyl-CoA, a gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding a 2-hydroxyalkanoyl-CoA, 3-hydroxy Hydroxybutyrate, 2-hydroxybutyrate and 2-hydroxybutyrate as a substrate, and a gene encoding a PHA synthetase using as a substrate, 4-hydroxybutyrate, 3-hydroxybutyrate and 2- As well as a method for producing the same.

In another example, a gene encoding lactate dehydrogenase is deleted, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate is converted to 2-hydroxyalkanoyl- Hydroxyalkanoyl-CoA, a gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding a 2-hydroxyalkanoyl-CoA, 3-hydroxy Hydroxybutyrate, 3-hydroxybutyrate, and 2-hydroxybutyrate, which comprises the step of introducing into a cell a gene encoding a PHA synthesizing enzyme that uses 4-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- There is provided a process for producing a microorganism which produces a terpolymer containing hydroxybutyrate as a repeating unit.

In one aspect, the present invention relates to a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as recurring units, and a process for its preparation.

A specific example provides a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as recurring units. For example, 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer.

Another example is that the activity of lactate dehydrogenase is weakened or deficient, the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is replaced with 3 -Hydroxyalkanoyl-CoA and encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding an enzyme that converts 2-hydroxyalkanoyl-CoA, 3-hydroxy Comprising the step of culturing a cell comprising a gene encoding a polyhydroxyalkanoate synthase using alkanoyl-CoA and 4-hydroxyalkanoyl-CoA as a substrate, 4-hydroxybutyrate, 3- Hydroxybutyrate and 2-hydroxybutyrate as repeating units.

In another aspect, the present invention relates to a microorganism producing a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, and a method for producing the same.

A specific example is that the activity of lactate dehydrogenase is weakened or deficient, the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate Hydroxyalkanoyl-CoA, and a gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding 2-hydroxyalkanoyl-CoA, 3- Comprising a gene encoding a PHA synthase using hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates and repeating 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate To a microorganism producing a terpolymer containing the unit as a unit.

In another example, a gene encoding lactate dehydrogenase is deleted, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate is converted to 2-hydroxyalkanoyl- Hydroxyalkanoyl-CoA, a gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding a 2-hydroxyalkanoyl-CoA, 3-hydroxy Hydroxybutyrate-2-hydroxybutyrate-2-hydroxybutyrate-2-hydroxybutyrate, which comprises the step of introducing into a cell a gene encoding a PHA synthesizing enzyme using as a substrate hydroxybenzoyl-CoA and 4-hydroxyalkanoyl- Hydroxybutyrate < / RTI > terpolymer.

Hereinafter, the configuration of the present invention will be described in more detail.

The term "terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units" means that the monomers 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate Refers to a linear polyester comprising repeating units polymerized by ester linkage. At this time, there is no particular limitation on the order of polymerization of each monomer, and it can be randomly repeated. Hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer, 3-hydroxybutyrate-2-hydroxybutyrate-4-hydroxybutyrate terpolymer, 2-hydroxybutyrate- Hydroxybutyrate-3-hydroxybutyrate terpolymer, 4-hydroxybutyrate-2-hydroxybutyrate-3-hydroxybutyrate terpolymer, 3-hydroxybutyrate-4-hydroxybutyrate- Butyrate terpolymer, 2-hydroxybutyrate-3-hydroxybutyrate-4-hydroxybutyrate terpolymer, and the like.

The term "2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoate Quot; is an enzyme that converts CoA from a CoA donor to 2-hydroxyalkanoate, 3-hydroxyalkanoate and 4-hydroxyalkanoate, respectively, to convert 2-hydroxyalkanoyl- -Hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA. Examples of the CoA donor include acetyl-CoA or acyl-CoA (e.g., propionyl-CoA).

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

For example, conversion of 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, conversion of 3-hydroxyalkanoate to 3-hydroxyalkanoyl-CoA, conversion of 4-hydroxyalkanoyl- The gene encoding an enzyme that converts et to 4-hydroxyalkanoyl-

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

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

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

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

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

(f) a base 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 the amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;

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

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

(j) a nucleotide sequence in which 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

≪ RTI ID = 0.0 > and / or < / RTI >

The term "PHA synthesizing enzyme using 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as a substrate" refers to 2-hydroxyalkanoyl-CoA, 3 -Hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates and synthesizing a terpolymer containing 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units Enzyme.

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

For example, the PHA synthesizing enzyme may be,

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

At least one mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R and A527S in the amino acid sequence of SEQ ID NO: And a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:

In another embodiment, the PHA synthetase is selected from the group consisting of:

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 nucleotide 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.

The enzymes may contain additional mutations within the scope of not totally altering the activity of the molecule. For example, amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. For example, the commonly occurring exchanges are carried out with amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / , Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly exchange. 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 against protein heat, pH, etc. is increased or protein activity is increased by mutation or modification of amino acid sequence.

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

The term "lactate dehydrogenase" refers to an enzyme that catalyzes the reversible conversion between pyruvate and lactate, and plays an essential role in the lactate synthesis pathway. In one embodiment, the gene encoding the lactate dehydrogenase may be ldhA.

In the present invention, lactate dehydrogenase, which is involved in the production of lactate during the metabolism of the host cell, is characterized in that the activity of the lactate dehydrogenase is weakened or deficient compared to the intrinsic control activity, in order to produce a lactate-free copolymer. The intrinsic regulatory activity means the active state of an enzyme that the host cell has in its natural state, and may mean, for example, an activity relating to the lactate synthesis naturally occurring in Escherichia coli.

The deletion of the lactate dehydrogenase activity can be carried out by genetic manipulation such as deletion or substitution of a part or all of the gene encoding the enzyme or insertion of a specific mutation sequence into the nucleotide sequence of the gene. Conventional knockout techniques known in the art can be applied at this time. In addition, the activity of lactate dehydrogenase may be weakened by modifying the base sequence of the expression regulatory sequence of the gene such as the promoter region or the 5'-UTR region of the gene to weaken the expression of the enzyme, By introducing a mutation at the site, the activity of the enzyme can be weakened. The introduction of such a mutation can be by any method known in the art, for example by homologous recombination, or by a lambda red recombination system.

The microorganisms provided herein convert 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, convert 3-hydroxyalkanoate to 3-hydroxyalkanoyl-CoA, A gene encoding an enzyme that converts a Roxy alkanoate to a 4-hydroxyalkanoyl-CoA, and a gene encoding 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- And a gene encoding a PHA synthase used as a substrate, and the genes may be introduced into cells by a recombinant method.

For example, the microorganism converts 2-hydroxyalkanoate to 2-hydroxyalkanoyl-CoA, converts 3-hydroxyalkanoate to 3-hydroxyalkanoyl-CoA, A gene encoding an enzyme that converts hydroxyalkanoate into 4-hydroxyalkanoyl-CoA, and a gene encoding 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl-CoA Which is used as a substrate, may be transformed with a recombinant vector or genetically engineered such that the gene is inserted on the chromosome.

In addition, the microorganism can be obtained by converting 2-hydroxyalkanoate into 2-hydroxyalkanoyl-CoA, converting 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA, A gene encoding an enzyme that converts alkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- And the other one may be transformed with a recombinant vector or genetically engineered such that the gene is inserted on the chromosome.

For example, the microorganism may be a cell comprising a gene encoding a PHA synthase using 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- , 2-hydroxyalkanoate was converted into 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate was converted into 3-hydroxyalkanoyl-CoA, 4-hydroxyalkanoate was converted into 3-hydroxyalkanoyl- May be obtained by transforming a gene encoding an enzyme that converts to 4-hydroxyalkanoyl-CoA.

As another example, the microorganism can be obtained by converting 2-hydroxyalkanoate into 2-hydroxyalkanoyl-CoA, converting 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA, Hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl-CoA are added to cells containing a gene encoding an enzyme that converts the Roxy alkanoate to 4-hydroxyalkanoyl- It may be one obtained by transforming a gene encoding PHA synthetase using a Nooll-CoA as a substrate.

Hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer is produced by a recombinant method or by using the microorganism to produce 4-hydroxybutyrate-3-hydroxybutyrate- The process for producing the hydroxybutyrate terpolymer may comprise the following steps.

First, 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoate is converted to 3- A gene encoding an enzyme that converts to 4-hydroxyalkanoyl-CoA, and a PHA using 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- And inserting at least one gene encoding a synthetic enzyme into a vector to prepare a recombinant vector. These two genes may be inserted into separate vectors or inserted into a single vector.

The term "vector" refers to a gene construct comprising an essential regulatory element operably linked to the expression of a gene insert encoding a desired protein in a cell of an individual, wherein the nucleic acid sequence encoding the desired protein is introduced into the host cell . Various vectors such as a plasmid, a virus vector, a bacteriophage vector, a cosmid vector, and a YAC (Yeast Artificial Chromosome) vector may be used as the above-mentioned vector. The recombinant vector includes a cloning vector and an expression vector. A cloning vector is a replicon that includes a replication origin, for example, a replication origin of a plasmid, phage or cosmid, and in which the fragment to which another DNA fragment is attached can be cloned. Expression vectors have been developed to be used to synthesize proteins.

The vector is not particularly limited insofar as it expresses the desired enzyme gene in various host cells such as prokaryotic cells or eukaryotic cells and is capable of producing it. However, the vector is inserted into the vector and the transferred gene is irreversibly fused into the genome of the host cell A vector that allows gene expression to be stably maintained in the cell for a long period of time is preferable.

Such vectors include transcriptional and detoxification expression control sequences that allow the gene to be expressed in a selected host. Expression control sequences may include promoters for conducting transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and / or sequences that control the termination of transcription and translation . For example, regulatory sequences suitable for prokaryotes may include a promoter, optionally an operator sequence and / or a ribosome binding site. Regulatory sequences suitable for eukaryotic cells may include promoters, terminators and / or polyadenylation signals. The initiation codon and the termination codon are generally considered to be part of the nucleic acid sequence encoding the target protein and should act in the individual when the gene construct is administered and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible. It may also contain a cloning source if it is a replicable expression vector. In addition, an enhancer, a toxin region at the 5 'end and a 3' end of the gene of interest, a selectable marker (for example, an antibiotic resistance marker), or a replicable unit may suitably be contained. The vector may be self-replicating or integrated into the host genomic DNA.

Examples of useful expression control sequences include early and late promoters of adenovirus, monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, long terminal repeat (LTR) promoter of HIV, Moloney virus, A lac system, a trp system, a human cytomegalovirus (CMV) promoter, an Epstein-Barr virus (EBV) promoter, a rosacekoma virus (RSV) promoter, an RNA polymerase II promoter, The TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the regulatory region of the fd coding protein, the promoter for phosphoglycerate kinase (PGK) or other glycolytic enzymes, the promoter of phosphatase , Such as Pho5, the promoter and prokaryotic cell of the yeast alpha-mating system, 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 detoxification expression control sequences should be operatively linked to each other. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. For example, if the DNA for a pre-sequence or secretory leader is expressed as a whole protein participating in the secretion of the protein, it can be operably linked to the DNA for the polypeptide and the promoter or enhancer Or the ribosome binding site can be operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site can be operably linked to the coding sequence if it affects the transcription of the sequence, , It can be operably linked to a coding sequence. The linkage of these sequences can be performed by ligation at convenient restriction sites and, if such sites are not present, using a synthetic oligonucleotide adapter or linker according to conventional methods . ≪ / RTI >

Those skilled in the art will appreciate that various vectors suitable for the present invention, expression controllability and the like, in consideration of the nature of the host cell, the number of copies of the vector, the ability to regulate the number of copies, and other proteins encoded by the vector, Sequence, and host can be selected.

Next, the microorganism is transformed using the recombinant vector.

The term "transformation" refers to the introduction of DNA into a host such that the DNA is replicable as an extrachromosomal element or by chromosome integration completion.

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 efficiency of introduction of DNA and high efficiency of expression of the introduced DNA can be usually 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 XL1-Blue), Escherichia spp., Pseudomonas spp., Bacillus spp., Streptomyces spp., Erwinia spp., Serratia spp., Providencia spp., Corynebacterium spp., Leptospira spp., Salmonella spp. But are not limited to, eukaryotic and prokaryotic hosts such as bacteria, hypomonas, chromobacterium, nocadia, fungi or yeast. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

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

Transformation methods include, but are not limited to, using appropriate standard techniques such as electroporation, electroinjection, microinjection, calcium phosphate co- precipitation, precipitation, calcium chloride / rubidium, retroviral infection, DEAE-dextran, cationic liposome, polyethylene glycol-mediated uptake, (gene gun), and the like, but the present invention is not limited thereto. At this time, the vector of circular form can be introduced into a linear vector form by cutting with appropriate restriction enzymes.

Next, the transformed microorganism is cultured to produce 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer.

The transformant expressing the recombinant vector can be cultured in a medium to produce and isolate a large amount of 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer. The medium and culture conditions can be suitably selected depending on the kind of transfected cells. The conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the terpolymer at the time of culturing. Examples of such culture methods include, but are not limited to, batch, continuous, and fed-batch cultivation.

In one embodiment, the culture may be performed in a medium comprising 2-hydroxybutyrate, 3-hydroxybutyrate and / or 4-hydroxybutyrate. Also, in the case of a microorganism capable of biosynthesizing 2-hydroxybutyrate, 3-hydroxybutyrate and 4-hydroxybutyrate from a carbon source such as glucose, 2-hydroxybutyrate, 3-hydroxybutyrate and / The copolymer can be prepared without adding the butyrate separately.

In addition to this, the medium used for cultivation should suitably satisfy the requirements of the specific strains. The medium may include 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, palmitic acid, Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of the nitrogen source in the medium include peptone, yeast extract, juice, 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, But is not limited thereto. The nitrogen source may also be used individually or as a mixture. Examples of the materials in the medium include, but are not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may include metal salts such as magnesium sulfate or iron sulfate necessary for growth, or may include essential growth materials such as amino acids and vitamins, but is not limited thereto. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

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 a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Oxygen or oxygen-containing gas (e.g., air) may be injected into the culture to maintain aerobic conditions, and the temperature of the culture may be usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The culture can continue until the desired yield of the desired terpolymer is obtained.

The next step is to recover the produced 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer.

The 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer produced from the recombinant microorganism can be isolated from the cell or culture medium in a manner well known in the art. Examples of the method for recovering the terpolymer include centrifugal separation, ultrasonic disruption, filtration, ion exchange chromatography, high performance liquid chromatography (HPLC), and gas chromatography (GC) The present invention is not limited to these examples.

The present invention provides a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units, which is a raw material for biodegradable and biocompatible bioplastics, Food, agricultural and medical fields.

Figure 1 shows the production process and cleavage map of the vector pPs619C1310-CpPCT540.
Fig. 2 shows a cleavage map of the vector pPs619C1249.18H-CPPCT540.
Fig. 3 shows the result of analysis of the 4-hydroxybutyrate-3-hydroxybutyrate-2-hydroxybutyrate terpolymer produced from the recombinant microorganism by gas chromatography.

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

Example  1. 4- Hydroxybutyrate -3- Hydroxybutyrate -2- Hydroxybutyrate  Preparation of recombinant vector for preparation of trimer

1-1. pPs619C1310 - CPPCT540  Preparation of recombinant vector

The propionyl-CoA transferase gene (pct) used a mutant of propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum, and the PHA synthase gene was Pseudomonas sp. MBEL 6-19 (KCTC 11027BP) were used. The vector used was 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'-CAG TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3 '(SEQ ID NO: 6)] And PCR was performed using the extracted whole 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 to _-19 expressing the synthase, pSYL105 vector (Lee et al . , Biotech. Bioeng ., 1994, 44: 1337-1347) in Ralstonia eutropha A pReCAB recombinant vector was prepared by digesting a DNA fragment containing the PHB-producing operon derived from H16 with BamHI / EcoRI and inserting it into the BamHI / EcoRI recognition site of pBluescript II (Stratagene Co., USA). The pReCAB vector contains the PHA synthase (phaC _RE ) and the monomeric donor enzyme (phaA _RE And phaB _RE ) are constantly expressed by the PHB operon promoter. BstBI / SbfI recognition site was removed BstBI each position and inherent priority to make phaC1 _{Ps6 -19} synthase gene fragments, one containing only the ends without changing the amino acid SDM (site directed mutagenesis) methods, BstBI / SbfI recognition (SEQ ID NO: 7), 5'-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC -3 '(SEQ ID NO: 8), 5'- 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' CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG -3 '(SEQ ID NO: 11), 5'-aac ggg agg gaa cct gca gg -3' (SEQ ID NO: 12)]. The pReCAB vector was digested with BstBI / SbfI to obtain 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 that affect SCL (short chain length) activity were found through amino acid sequence analysis. The primers [5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- 3 ' - 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- 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) for using the SDM method using, E130D, S325T, phaC1 _{Ps6 -19} Q481M containing a pPs619C1300- containing synthase mutant of phaC1 _{Ps6 -19} 300 ReAB was prepared.

In order to construct an always expressed system of the operon type in which the propionyl-CoA transferase is expressed in the same manner, propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum Respectively. CP-PCT was constructed by amplifying the chromosomal DNA of Clostridium propionikum with the primers [5'-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3 '(SEQ ID NO: 19), 5'-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg- (SEQ ID NO: 20)] was used. At this time, the NdeI site existing in the original wild-type CP-PCT was removed by SDM method for ease of cloning, and the primer [5'-agg cct gca ggc gga taa caa ttt cac 3 '(SEQ ID NO: 21), 5'-gcc catgatte agatta gga cttcat ttc c-3' (SEQ ID NO: 22). The pPs619C1300-ReAB vector was digested with SbfI / NdeI to obtain Ralstonia eutrophus The H16-derived monomer-supplying enzyme (phaA _RE And phaB _RE ), and then the PCR-cloned CP-PCT gene was inserted into the SbfI / NdeI recognition site to prepare a pPs619C1300-CPPCT recombinant vector.

Next, primers [5'-CGCCGGCAGGCCTGCAGG-3 '(SEQ ID NO: 23), 5'-GGCAGGTCAGCCCATATGTC (SEQ ID NO: 23)) were prepared using the above prepared pPs619C1300-CPPCT as a template to introduce a random mutagenesis into the CP- -3 '(SEQ ID NO: 24) was used to perform error-prone PCR under the condition that Mn ^{2 +} was added and the concentration difference of dNTPs existed. Thereafter, PCR was carried out under normal conditions using the above primers to amplify a PCR fragment containing a random mutation. pPs619C1300-CPPCT then by cutting the vector with SbfI / NdeI to remove wild-type CPPCT, to create the ligation mixture was inserted into the amplified PCR fragments to the mutant SbfI / NdeI recognition site introduced in E. coli JM109 ~ 10 ⁵ degree scale Of CP-PCT library. The prepared CP-PCT library was grown in a polymer detection medium (LB agar, glucose 20 g / L, 3HB 1 g / L, Nile red 0.5 μg / ml) for 3 days, Candidates of 80 individuals were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ℃) for 4 days under the conditions of polymer production and analyzed by FACS (Florescence Activated Cell Sorting) 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. Various CP-PCT variants were obtained by random mutagenesis using the above-mentioned error-prone PCR method based on the above-mentioned primary mutants (CP-PCT Variant 512 and CP-PCT Variant 522) The CP-PCT Variant 540 (including Val193Ala and the silent mutants T78C, T669C, A1125G, and T1158C) was secondly selected 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 at c cc gcg gct cg cag cac gaa ttc- 3 '(SEQ ID NO: 26), 5'-ggg cat atc aag agc atc ctg aac ccg c-3' (phaC1 _{Ps6 -19} 310) derived from Pseudomonas sp. MBEL 6-19 having the amino acid sequence of E130D, S477F and Q481K mutated using the SDM method using the cct gat atg ccc-3 '(SEQ ID NO: 28) (PPs619C1310-CPPCT540 vector) (Fig. 1).

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

PCR was performed using the 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 above as a template. Respectively. After performing error-prone PCR, PCR was performed again using the above primers to amplify the PCR fragment containing the mutation, and the amplified mutants were inserted into the BstBI / SbfI site of the pPs619C1310-CPPCT540 vector to construct a library of mutants Respectively. The prepared mutant library was transformed into E. coli XL-1 Blue and cultured in PHB detection medium (LB agar, glucose 20 g / L, Nile red 0.5 μg / ml) for 3 days. The final screened mutants through screening after incubation were pPs619C1249.18H with the mutated amino acid sequence of 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. ldhA  The gene Knock-out  E. coli  XL1- Blue Mutant  making

In order to produce a lactate-free polymer based on Escherichia coli XL1-Blue (Stratagene, USA), D-lactate dehydrogenase (LdhA) involved in lactate production during the metabolism of E. coli was transformed into genomic DNA Knocked out. Genetic deletions were made using the red-recombination method well known in the art. The oligomer used to delete ldhA was synthesized by the nucleotide sequence of SEQ ID NO: 31 (5'-atcagcgtacccgtgatgctaacttctctctggaaggtctgaccggctttaattaaccctcactaaagggcg-3 ') and SEQ ID NO: 32 (5'-atcagcgtacccgtgatgctaacttctctctggaaggtctgaccggctttaattaaccctcactaaagggcg-3').

Example  3. 4- Hydroxybutyrate -3- Hydroxybutyrate -2- Hydroxybutyrate Of the terpolymer  Produce

The recombinant vector prepared in Example 1 was transformed into E. coli XL1-BlueΔldhA knock-out of ldhA prepared in Example 2 using electroporation to produce recombinant E. coli XL1-BlueΔldhA . Flask culture was carried out to prepare the above terpolymer. First, for the seed culture, the recombinant E. coli was inoculated into 3 mL of LB medium [Bacto ^™ Triptone (BD) 10 g / L, Bacto ^™ yeast extract (BD) containing 100 mg / L ampicillin and 20 mg / L kanamycin (BD) 5 g / L, NaCL (amresco) 10 g / L] for 12 hours. For this cultivation, 1 ml of the preculture was added to 1 g / L 4-hydroxybutyrate (4-HB), 1 g / L 3-hydroxybutyrate (3-HB), 1 g / L 2-hydroxybutyrate (10 g Glucose per liter, 6.67 g KH ₂ PO ₄ , ₄ g NH ₄ ) 2HPO ₄ , 10 g / L thiamine, 20 mg / L kanamycin and 10 mg / L thiamine, _{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 per _{1L 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) 6Mo 7 O 2 · 4H 2 O 0.1g, and _{Na 2 B 4 O 2 · 10H} 2 O 0.02g) And cultured at 30 DEG C for 3 days with stirring at 250 rpm.

The culture was centrifuged at 4,000 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. The microbial cells were quantified and reacted with methanol under a sulfuric acid catalyst using chloroform as a solvent at 100 ° C. The mixture was added at room temperature with distilled water of half the volume of chloroform, mixed and allowed to stand until separated into two layers. The chloroform layer in which the monomers of the methylated polymer were dissolved was collected from the two layers, and the components of the polymer were analyzed by gas chromatography (GC). As the internal reference material, benzoate was used. The GC analysis conditions used at this time are shown in Table 1 below.

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

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

Total PHA content (wt%) Polymer (mol%) 4HB 3HB 2HB 9.30 18.5 38.8 42.7

<110> LG CHEM, LTD. Copolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and          2-hydroxybutyrate as repeating unit and method for preparing the          same <130> DPP20147706KR <160> 32 <170> Kopatentin 1.71 <210> 1 <211> 1575 <212> DNA <213> Clostridium propionicum <220> <221> gene &Lt; 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 cagaaaaaaat 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 &Lt; 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 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 Gly 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 Aly Gly Aly Gly Aly Gly Gly Val Ala Gly Gly Val                 325 330 335 Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr             340 345 350 Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly         355 360 365 Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly     370 375 380 Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr 385 390 395 400 Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val                 405 410 415 Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys             420 425 430 Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala         435 440 445 Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe     450 455 460 Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile 465 470 475 480 Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile                 485 490 495 Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe             500 505 510 Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser         515 520 <210> 3 <211> 1677 <212> DNA <213> Pseudomonas sp. 6-19 <220> <221> gene <222> (1) .. (1677) <223> PHA synthase <400> 3 atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360 gggcacttcg tgatcaacct catgaccgaa gcgatggcgc cgaccaacac cgcggccaac 420 ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720 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 <213> Pseudomonas sp. 6-19 <220> <221> PEPTIDE &Lt; 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 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 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 <210> 31 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> oligomer <400> 31 atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt aattaaccct 60 cactaaaggg cg 72 <210> 32 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> oligomer <400> 32 atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt aattaaccct 60 cactaaaggg cg 72

Claims (15)

4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units.
The activity of lactate dehydrogenase is weakened or deficient, the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, and the 2-hydroxyalkanoyl- 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoate is converted into 4-hydroxyalkanoate. A gene encoding an enzyme that converts to 4-hydroxyalkanoyl-CoA, and 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl-CoA as substrates Comprising the step of culturing a microorganism comprising a gene encoding a polyhydroxyalkanoate (PHA) synthase,
4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units.
The microorganism according to claim 2, wherein the microorganism converts 2-hydroxyalkanoate into 2-hydroxyalkanoyl-CoA, converts 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA, A gene encoding an enzyme that converts 4-hydroxyalkanoate to 4-hydroxyalkanoyl-CoA, and a gene encoding 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA, and 4-hydroxyalkanoyl Which is obtained by transforming a gene encoding PHA synthetase using -CoA as a substrate.
The method of claim 2, wherein the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, Wherein the enzyme converting the Roxy alkanoate to 4-hydroxyalkanoyl-CoA is propionyl-CoA transferase.
The method of claim 2, wherein the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, The gene encoding the enzyme that converts the Roxy alkanoate to 4-hydroxyalkanoyl-
(a) a nucleotide sequence of SEQ ID NO: 1;
(b) a base sequence in which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(c) a base sequence in which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(d) a base sequence in which Gly335Asp is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(e) a base sequence in which Ala243Thr is mutated in the amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(f) a base 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 the amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(h) a nucleotide sequence in which Asp65Asn is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(i) a base sequence in which Thr199Ile is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1; And
(j) a nucleotide sequence in which 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
&Lt; / RTI &gt;
The production method according to claim 2, wherein the polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Pseudomonas sp. 6-19.
3. The polyhydroxyalkanoate synthase according to claim 2, wherein the gene encoding the polyhydroxyalkanoate-
The amino acid sequence of SEQ ID NO: 4; or
At least one mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R and A527S in the amino acid sequence of SEQ ID NO: &Lt; / RTI &gt; wherein the nucleotide sequence is a nucleotide sequence corresponding to the amino acid sequence.
3. The polyhydroxyalkanoate synthase according to claim 2, wherein the gene encoding the polyhydroxyalkanoate-
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 nucleotide 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.
3. The process according to claim 2, wherein the culture is carried out in a medium comprising 2-hydroxybutyrate, 3-hydroxybutyrate and 4-hydroxybutyrate.
The activity of lactate dehydrogenase is weakened or deficient,
Hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, 4-hydroxyalkanoate is converted to 4- A gene encoding an enzyme that converts to hydroxyalkanoyl-CoA, and a PHA synthetase using 2-hydroxyalkanoyl-CoA, 3-hydroxyalkanoyl-CoA and 4-hydroxyalkanoyl- Lt; RTI ID = 0.0 &gt;
A microorganism producing a terpolymer comprising 4-hydroxybutyrate, 3-hydroxybutyrate and 2-hydroxybutyrate as repeating units.
11. The method of claim 10, wherein the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, The enzyme that converts Roxy alkanoate to 4-hydroxyalkanoyl-CoA is a propionyl-CoA transferase, a microorganism.
11. The method of claim 10, wherein the 2-hydroxyalkanoate is converted to 2-hydroxyalkanoyl-CoA, the 3-hydroxyalkanoate is converted to 3-hydroxyalkanoyl-CoA, The gene encoding the enzyme that converts the Roxy alkanoate to 4-hydroxyalkanoyl-
(a) a nucleotide sequence of SEQ ID NO: 1;
(b) a base sequence in which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(c) a base sequence in which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(d) a base sequence in which Gly335Asp is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(e) a base sequence in which Ala243Thr is mutated in the amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(f) a base 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 the amino acid sequence corresponding to SEQ ID NO: 1 and A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;
(h) a nucleotide sequence in which Asp65Asn is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1;
(i) a base sequence in which Thr199Ile is mutated in the amino acid sequence corresponding to SEQ ID NO: 1, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1; And
(j) a nucleotide sequence in which 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
&Lt; / RTI &gt; wherein the nucleotide sequence is selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
11. The microorganism according to claim 10, wherein the polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Pseudomonas sp. 6-19.
11. The method according to claim 10, wherein the gene encoding the polyhydroxyalkanoate synthase is selected from the group consisting of:
The amino acid sequence of SEQ ID NO: 4; or
At least one mutation selected from the group consisting of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, Q481R and A527S in the amino acid sequence of SEQ ID NO: Wherein the nucleotide sequence is a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:
11. The method according to claim 10, wherein the gene encoding the polyhydroxyalkanoate synthase is selected from the group consisting of:
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 nucleotide 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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