WO2012124941A2

WO2012124941A2 - Recombinant microorganism having ability to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer, and method for preparing polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer using same

Info

Publication number: WO2012124941A2
Application number: PCT/KR2012/001752
Authority: WO
Inventors: 이상엽; 박시재; 이승환; 송봉근; 이태우
Original assignee: 한국과학기술원; 한국화학연구원
Priority date: 2011-03-11
Filing date: 2012-03-09
Publication date: 2012-09-20
Also published as: KR101328567B1; KR20120103997A; WO2012124941A9; WO2012124941A3

Abstract

The present invention relates to a polyhydroxyalkanoate containing a 2-hydroxybutyrate as a monomer, a recombinant microorganism having the ability to produce the polyhydroxyalkanoate containing the 2-hydroxybutyrate as the monomer, and a method for preparing the polyhydroxyalkanoate containing the 2-hydroxybutyrate as the monomer. The present invention can effectively provide the polyhydroxyalkanoate containing the 2-hydroxybutyrate, which is a novel biodegradable polymer, and the recombinant microorganism for producing same.

Description

Recombinant microorganism having the ability to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer and a method for producing polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer

The present invention provides a recombinant microorganism having the ability to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer, polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer, and 2 It relates to a method for producing polyhydroxyalkanoate containing -hydroxybutyrate as a monomer.

Polyhydroxyalkanoates (PHAs) are biodegradable, biocompatible polyesters composed of several types of hydroxy-carboxylic acids used in bacteria as carbon sources and / or reducing storage materials. PHA is synthesized by PHA synthase, which uses several kinds of hydroxyacyl-CoA as a substrate.

If hydroxyacyl-CoA has an asymmetric center at the carbon position of the hydroxy group, it is all an (R) -configuration. Among the various hydroxyacyl-CoAs, such as 3-hydroxyacyl-CoAs, 4-hydroxyacyl-CoAs, 5-hydroxyacyl-CoAs, 6-hydroxyacyl-CoAs, and the like, PHA synthase is the most preferred substrate. Roxiasil-CoAs (3HA-CoAs).

However, there have been no reports of producing PHAs containing 2-hydroxy acids as monomers in microorganisms producing PHAs. Natural PHA synthase has a negligible level of activity against 2-hydroxyacyl-CoA, much lower than other hydroxyacyl-CoAs.

Recently, we have developed a metabolic recombinant E. coli that can produce polylactic acid (PLA) polymers and polymers thereof by fermentation directly from glucose (WO08 / 062996, 2008; Yang et al., Biotechnol. Bioeng. 105 , 150-160 , 2010; Jung et al., Biotechnol. Bioeng. 105 , 161-171, 2010).

In the recombinant Escherichia coli, PLA biosynthesis consists of two stages: lactyl-CoA production and copolymerization by synthase. (D) -Lactyl-CoA is a propionyl-CoA transferase of Clostridium propionicum which converts (D) -lactate to (D) lactyl-CoA using acetyl-CoA as a CoA donor in the recombinant E. coli. CoA transferase) was employed to enable production. To produce lactate polymer (PLA) with lactyl-CoA, Pseudomonas sp. Was genetically engineered to use lactyl-CoA as a substrate. 6-19 PHA synthase (PhaC1 _ps6-19 ) was employed. In addition, in order to increase the efficiency of PLA synthesis, the activity of propionyl-CoA transferase of Clostridium propionicum was enhanced by random denaturation (randum mutageneisis) in E. coli XL1-Blue to improve lactyl-CoA biosynthesis ability. (Yang et al., Biotechnol. B ioeng. 105 , 150-160 , 2010; Jung et al., Biotechnol. Bioeng. 105 , 161-171, 2010).

In other studies, reports have also been made of biosynthesis of (3-hydroxybutyrate-co-lactate) polymer [P (3HB-co-LA)] using a similar system (Taguchi et al., Proc. Natl. Acad. Sci. USA. 105 , 17323-17327, 2008, Yamadae et al., Biomacromolecules 10 , 677-681, 2009). Since PhaC1 _ps6-19 has a wide variety of substrate specificities and lactate is one type of 2-hydroxy acid, another 2-hydroxyacyl-CoA is used as the substrate of PhaC1 _ps6-19 It will also be possible to synthesize new PHAs that contain. Among these various kinds of 2-hydroxy acids, the present inventors attempted polymer synthesis using 2-hydroxybutyrate (2HB), which is a metabolite of the amino acid synthesis pathway, as a monomer. Recently, a citramalate pathway that produces 1-propanol and 1-butanol and converts pyruvate to 2-ketobutyrate has been developed by employing citramalate synthase (CimA) of Methanococcus jannaschii .

Accordingly, the present inventors have diligently developed a new PHA, and as a result, express the 2-hydroxybutyrate dehydrogenase gene to produce 2-hydroxybutyl-CoA, which is a substrate of PhaC1 _ps6-19 , and the citramalate pathway. When a recombinant Escherichia coli is prepared by metabolizing E. coli to express propionyl-CoA transferase gene, and fermenting the recombinant Escherichia coli, a new polymer, 2-hydroxybutyrate-co-3-hydroxybutyrate) polymer It was confirmed that [P (2HB-co-3HB)] could be prepared and the present invention was completed.

Summary of the Invention

An object of the present invention is to provide a polyhydroxyalkanoate containing 2-hydroxybutyrate, a biodegradable novel polymer as a monomer, and a recombinant microorganism producing the polymer.

In order to achieve the above object, the present invention is a gene encoding lacdate dehydrogenase is deleted, a gene encoding a polyhydroxy alkanoate synthase and a gene encoding propionyl-CoA transferase is introduced The present invention provides a recombinant microorganism having a polyhydroxyalkanoate producing ability containing 2-hydroxybutyrate as a monomer.

The present invention also provides a step of culturing the recombinant microorganism in a medium containing 2-hydroxybutyrate and 3-hydroxybutyrate to produce (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer. ; And obtaining (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer produced above. Tate) polymer is provided.

The present invention also provides a gene encoding a lacdate dehydrogenase, a gene encoding a polyhydroxy alkanoate synthase and a gene encoding propionyl-CoA transferase, a D-2 hydroxy acid. A gene encoding dehydrogenase, a gene encoding simalate synthase, and a gene encoding 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate / (R) -2-methylmalate dehydratase enzyme (leuBCD) are introduced. To provide a recombinant microorganism having the ability to produce (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate).

The present invention also comprises the steps of culturing the recombinant microorganism in a medium containing glucose and 3-hydroxybutyrate to produce a (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer; And obtaining (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer produced above. Tate) polymer is provided.

The present invention also provides a (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer.

Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.

1 shows a biosynthetic pathway for synthesizing polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer from glucose and 3HB.

Figure 2 shows the results of analyzing the composition and sequence contribution for the P (2HB-co-3HB) prepared in the present invention by 1D ( ¹ H and ¹³ C) NMR and 2D ( ¹ H- ¹ H) COSY NMR spectrum will be.

Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In view of the above, the present invention includes a gene encoding lacdate dehydrogenase, a gene encoding a polyhydroxy alkanoate synthetase and a gene encoding propionyl-CoA transferase, The present invention relates to a recombinant microorganism having a polyhydroxyalkanoate producing ability containing 2-hydroxybutyrate as a monomer.

In the present invention, the polyhydroxy alkanoate synthetase is Pseudomonas sp. PHA synthase or Pseudomonas sp. It may be characterized by the mutation of the PHA synthase of 6-19.

In the present invention, the gene encoding the mutant of the PHA synthase is selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 1 It may be characterized in that it has a base sequence corresponding to the amino acid sequence comprising one or more mutations, wherein the gene encoding the mutase of PHA synthase is E130D, S325T, L412M, S477G and Q481M in the amino acid sequence of SEQ ID NO: 12 This mutated amino acid sequence (C1335); Amino acid sequence (C1310) in which E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 12; And in the amino acid sequence of SEQ ID NO: 12 E130D, S477F and Q481R may be characterized by having a base sequence corresponding to any one amino acid sequence selected from the group consisting of mutated amino acid sequence (C1312).

In the present invention, the propionyl -CoA transferase may be characterized in that the mutant enzyme of the transferase or propionyl propionyl -CoA -CoA transferase of C. propionicum of C. propionicum Pct540.

We have improved the productivity of p (3HB-co-LA) using recombinant E. coli expressing mutated PhaC1 _ps6-19 and mutated Pct _cp .

The mutated PhaC1 _ps6-19 has various substrate specificities, lactate also because it is a type of 2-hydroxy acid, the 2HB-CoA by PhaC1 _ps6-19 may be copolymerized in 2HB-containing polymer. To facilitate the synthesis of 2HB containing polymers in recombinant E. coli, 3HB was chosen as the second monomer because 3-HB-CoA acts as an initiator in the synthesis of lactate containing polymers.

In previous studies by the present inventors, the variation PhaC1 _ps6-19 and mutated Pct _{cp ,,} in Pct _cp has a V193A mutation and four silent mutations of the nucleotide sequence (T78C, T669C, A1125G and T1158C) and Pct540 PhaC1 _ps6-19d Recombinant Escherichia coli containing PhaC1437 containing four variants of E130D, S325T, S477G and Q481K in E. coli efficiently produced P (3HB-co-LA) copolymer with high lactate fraction. Thus, these enzymes were used for the biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate as monomer.

In the present invention, in order to inhibit the lactate monomer from being incorporated into the polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer, a host cell in which the ldhA gene was deleted was used.

In one embodiment, the present invention is a (2-hydroxybutyrate-co-3-hydroxybutyrate-co-lactate) polymer when the recombinant microorganism is incubated in a medium containing 2-hydroxybutyrate and 3-hydroxybutyrate Can produce

In another aspect, the invention provides a gene encoding a lacdate dehydrogenase, a gene encoding a polyhydroxy alkanoate synthetase and a gene encoding propionyl-CoA transferase, D-2 hydrate. Genes encoding hydroxy acid dehydrogenase, genes encoding simaleate synthase and genes encoding 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate / (R) -2-methylmalate dehydratase enzyme (leuBCD) The present invention relates to a recombinant microorganism having a polyhydroxyalkanoate-producing ability, in which 2-hydroxybutyrate is introduced and containing as a monomer.

In the present invention, the polyhydroxy alkanoate synthetase is Pseudomonas sp. PHA synthase or Pseudomonas sp. It may be characterized in that PhaC1 _p which is a mutant enzyme of PHA synthase of 6-19, the propionyl-CoA transferase is propionyl-CoA transferase of C. propionicum or propionyl-CoA transferase of C. propionicum It may be characterized in that the mutation is Pct540.

In the present invention, the D-2 hydroxy acid dehydrogenase may be characterized in that it is derived from Lactobacillus lactis , the simalate synthase may be characterized in that derived from Methanococcus jannaschii , the 2-isopropylmalate synthase , 3-isopropylmalate dehydrogenase, 3-isopropylmalate / (R) -2-methylmalate dehydratase enzymes may be characterized as being derived from E. coli.

In the present invention, the gene encoding the PHA biosynthetic gene transcription terminator may be further introduced, and the PHA biosynthetic gene transcription terminator may be derived from Ralstonia eutropha .

In another aspect, the present invention comprises the steps of culturing the recombinant microorganism in a medium containing glucose and 3-hydroxybutyrate to produce a polyhydroxy alkanoate containing 2-hydroxybutyrate as a monomer; And a method for producing polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer, comprising the step of obtaining polyhydroxy alkanoate containing (2-hydroxybutyrate as a monomer). It is about.

In an embodiment of the present invention, E.coli XL1-Blue polyhydroxy al, which contains the E. coli strain XLdh removal of the ldhA gene in chromosome 2-hydroxybutyrate as the monomer for the decanoate of P (co-2HB -3HB) was used as a host cell for copolymer synthesis.

The inventors have tried to employ a 1-propanol synthesis route using 2-ketobutyrate as a precursor because 2-ketobutyrate can be used as a precursor of 2-HB, producing the 2-ketobutyrate. The two pathways, the threonine degradation pathway and the citramalate pathway, the citratelate pathway that most directly produces 2-ketobutyrate were employed.

In one embodiment of the present invention, in order to establish the 2-ketobutyrate synthesis pathway, the expression of cimA3.7 gene of Methanococcus jannaschii and leuBCD gene of E. coli in the host cell through gene recombination.

In order to establish a synthetic route of the polymer containing 2HB from glucose in the present invention, the 2-ketobutyrate synthesized through the citramalate pathway must be converted to 2HB.

Some microbial D-2-hydroxy acid dehydrogenases have selectivity for the optical isomers and use NADH as a cofactor to reduce 2-keto acid to D-2-hydroxy acid. . The D-2-hydroxy acid dehydrogenase is 2-ketoisocaproate, 2-ketoisovalerate, 2-ketovalorate, 2-ketobutyrate and It has a wide range of substrate specificities, including mandelate.

In one embodiment of the invention, L. lactis subsp. 2HB was synthesized by employing two D-2 hydroxy acid dehydrogenases encoded by panE gene of lactis Il1403 and C. difficile 630 ldhA gene, and recombinant E. coli transformed with C. difficile 630 ldhA gene produced 2HB. Since no polymers were contained, the production of 2HB-containing polymers was subsequently carried out in L. lactis subsp. Recombinant E. coli transformed with the panE gene of lactis I 11403 was used.

L. lactis subsp. to recombinant E. coli XLdh expressing phaC437, pct540, cimA3.7 and leuBCD genes . When introducing the panE gene of lactis Il1403, more than 18.9% by weight of P (97 mol% 2 HB-co-3 mol% LA) polymer could be prepared from glucose.

In one embodiment of the present invention, when synthesizing p (2HB-co-3HB) polymer using recombinant E. coli, 2HB must be produced intracellularly from glucose, and 2HB is converted to 2HB-CoA by Pct540. do. In order to produce 3HB-CoA in cells, 3HB had to be added to the medium, and as the concentration of 3HB in the medium increased, the 2HB monomer fraction in the synthesized polymer decreased.

In another aspect, the present invention comprises the steps of culturing the recombinant microorganism in a glucose-containing medium to produce a polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer; And it provides a method for producing a polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer comprising the step of obtaining a polyhydroxyalkanoate containing the produced 2-hydroxybutyrate as a monomer. do.

In the present invention, when culturing the recombinant microorganism of the present invention in a glucose-containing medium, 2-hydroxybutyrate is produced by 2-hydroxybutyrate monomer by copolymerization of a monomer and 3-hydroxybutyrate monomer produced by the acetoacetyl CoA pathway. The p (2HB-co-3HB) polymer which is polyhydroxyalkanoate containing a butyrate as a monomer can be manufactured.

In another aspect, the present invention relates to a (2-hydroxybutyrate-co-3-hydroxybutyrate) polymer.

In the present invention, it was confirmed that amorphous P (2HB-co-3HB) was synthesized by recombinant E. coli, and the molecular weight of P (2HB-co-3HB) was 20,000 (Mn) and 33,800 (Mw) having 1.69 PDI. . Glass phase transition temperature was 9.7 degreeC.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Table 1 shows the strains and plasmids used in the examples below.

Table 1

Example 1: Biosynthesis of (2-hydroxybutyrate-co-3-hydroxybutyrate) polymer [P (2HB-co-3HB)] from 2HB and 3HB using recombinant strains

In this embodiment, the lactate monomer is P (3HB-co-2HB) of Pseudomonas sp mutations in the host cell of the ldhA gene deletion in order to inhibit the incorporation of the copolymer. Recombinant strains to which 6-19 PHA synthase and mutated C. propionicum propionyl-CoA transferase were introduced to prepare P (2HB-co-3HB) in a medium containing 2HB and 3HB.

E. coli XL1-Blue (Stratgene Cloning System, USA) was used as a host cell for gene cloning, and cultured in 37 ° C. LB medium.

In order to suppress the incorporation of lactate monomer into the P (2HB-co-3HB) copolymer, one-step inactivation of the ldhA gene on the chromosome of E. coli XL1-Blue (Datsenko and Wanner, Proc. Natl. Acad. Sci USA, 97 , 6640-6645, 2000), the E. coli XLdh strain was removed as a host cell for P (2HB-co-3HB) synthesis (Fig. 1).

E. coli XLdh strain was incubated for 72 hours at 30 rpm at 250 rpm using MR medium containing 20 g / L glucose and desired concentrations of 2HB and 3HB, and the concentration of 2HB and 3HB was controlled according to the culture conditions. It was.

Genes involved in 2-hydroxybutyl-CoA production include E. coli W3110 (KCTC), Clostridium difficile 630 (ATCC), and Lactococcus lactis subsp. Amplified from chromosomal DNA and plasmid p619C1437-pct540 (Yang et al ., Biotechnol. Bioeng. 105 , 150-160, 2010) of lactis Il1403 (ATCC).

Plasmid p619C1437-pct540 was prepared under the PHA synthase operon promoter of Ralstonia eutropha , Pseudomonas sp. PhaC1437 containing four variants of E130D, S325T, S477G, and Q481K in 6-19 PHA synthase and four silent mutations of V193A and nucleotide sequences in propionyl-CoA transferase of C. propionicum (T78C, T669C, A1125G) And T1158C), a recombinant plasmid expressing Pct540 (Yang et al ., Biotechnol . Bioeng. 105,150-160,2010).

Expression of the recombinant gene in recombinant E. coli XLdh was performed by adding IPTG to a concentration of 1 mM when the absorbance of the culture medium reached 0.5 at 600 nm.

Recombinant E. coli XLdh strains expressing PhaC1437 and Pct540 were incubated for 72 hours at 30 ° C. and 250 rpm using MR medium containing 20 g / L glucose and desired concentrations of 2HB and 3HB, depending on the culture conditions. The concentrations of 2HB and 3HB were adjusted.

Recombinant E. coli XLdh strains expressing PhaC1437 and Pct540 produced P (2HB-co-3HB) random copolymers containing various fractions of 2HB monomer in the range of 10 mol% to 60 mol% (Table 2).

TABLE 2

By controlling the concentration of 2HB and 3HB in the medium was able to control the mole fraction of 2HB in the copolymer, the mole fraction of 2HB in the copolymer was inversely proportional to the concentration of 2HB and 3HB added to the medium.

The amount of polymer increased in proportion to the total concentration of 2HB and 3HB added. When 2HB and 3HB were added at concentrations of 2 g / L, respectively, the amount of polymer produced was the most at 65% by weight.

Example 2: Biosynthesis of P (2HB-co-3HB) from Glucose Using Recombinant Strains

In this example, the recombinant E. coli XLdh expressing phaC437, pct540, cimA3.7 and leuBCD genes was expressed in L. lactis subsp. p (2HB-co-3HB) was prepared from glucose by introducing panE gene of lactis Il1403.

The primers used in the preparation of the recombinant plasmid in this example are shown in Table 3.

TABLE 3

The expression vector pZE12-MCS was modified to have muti-cloning sites (MCS) of pTacLac (Lee et al. Appl. Microbiol. Biotechnol. 79,633-641,2008) and a PHA biosynthetic gene transcription terminator of Ralstonia eutropha .

First, a PHA biosynthetic gene transcription terminator gene fragment of Ralstonia eutropha was obtained by PCR from p619C1437-pct540 using the primers of SEQ ID NOs: 1-2, using the primers of SEQ ID NOs: 6-5 using the obtained gene fragment as a template. PCR was performed to obtain PHA biosynthetic gene transcription terminators of RBS, pTacLac MCS and R. eutropha . The PCR product was digested with MfeI and AvrII and inserted into pZE12-MCS digested with EcoRI / AvrII to prepare pKE12-MCS.

by substituting pKE31-MCS (EXPRESSYS) _LtetO of _P-1 promoter in a P _LlacO-1 promoter obtained by cutting the pKE12-MCS with XhoI / EcoRI were prepared pKA32-MCS.

p619C1437 LeuBCD amplifies the E. coli leuBCD gene by PCR using primers SEQ ID NOs: 4-5, inserts the amplification product into the sbfI and NdeI sites of the template plasmid, and inserts the E.coli leuBCD gene into the pct540 gene of p619C1437-pct540. It was prepared by replacing with.

By inserting a synthetic gene into the EcoRI and cimA3.7 sbfI portion of the MCS-pKA32, it was prepared pKA32-CimA.

PKA32-CimALeuBCD was prepared by inserting the E. coli leuBCD gene obtained by cleaving the p619C1437 LeuBCD with sbfI and HindIII into the pKA32-CimA.

The pKA32-MCS was PCR using primers of SEQ ID NOs: 6 to 7 to amplify a gene fragment including the P _LlacO-1 promoter, MCS and PHA biosynthetic gene transcription terminator of R. eutropha, and the amplified gene fragment was amplified. pKM22-MCS was prepared by inserting into the SspI site of pBR1MCS2 ( Kovachetal. Gene , 166, 175-176, 1995).

to the pKM22-MCS insert lactisIl1403panE gene of L. lactis, we were prepared pKM22PanE, by inserting the gene of C.difficile 630ldhA in pKM22, was prepared pKM22LdhA.

In addition, the M.jannaschii cimA3.7 gene was used to synthesize based on the sequence shown in GenScript (www.genscript.com). Plasmids pZE12-MCS and pZA31-MCS were purchased from ESPRESSYS (www.expressys.com) and received.

Recombinant E. coli XLdh transformed with the plasmid expressing the phaC437, pct540, cimA3.7 , leuBCD and panE genes produced at least 18.9% by weight P (97 mol% 2 HB-co-3 mol% LA) polymer from glucose. (Table 4).

As the concentration of 3HB in the medium was increased, the 2HB monomer fraction in the synthesized polymer decreased.

Table 4

Example 3: Analysis of Polymers Synthesized in Recombinant Strains

The exemplary polymer prepared in Example 2 was isolated by extraction (Jacquel et al., Biochem. Eng. J. 39, 15-27,2008) cell from the organic solvent. The structure, molecular weight and thermal properties of the polymers were determined by NMR, gel chromatography (GPC) and diffrential scanning calorimetry (DSC), respectively (Yang et al., Biotechnol. Bioeng. 105 , 150-160 , 2010; Jung et al., Biotechnol. Bioeng. 105 , 161-171, 2010; Limetal., J. Mol. Struct. 886 , 166-174, 2008).

P (3HB-co-2HB), the composition and sequence contribute to 1D ⁽¹ H and ¹³ C) NMR and 2D ^⁽¹ H- ¹ H) were analyzed by COSYNMR spectrum (A ~ C shown in FIG. 2). The mole fractions of 2HB and 3HB were analyzed by ¹ H NMR and reconfirmed by GC analysis. P (2HB-co-3HB) 1H NMR spectrum in CDC ₁₃ is shown in A of FIG. 2. Methyl protons of 2HB were found in the δ0.7-1.2ppm section, and methylene protons were found in the δ1.6-2.1 ppm section. Methylene protons of 3HB were found in the δ 2.3-2.9 ppm range. On the other hand, oxymethine quantum of 2-HB-co-3HB was found at δ4.5-5.7ppm.

The COZY spectrum results are shown in B of FIG. 2, and the signal ① between both the oxymethine of 2HB and the methylene of 2HB was shown at δ4.9 ppm / 2.0 ppm. Coupling ② between 2HB methylene protons and 2HBdml methyl protons appeared as cross peaks at δ1.8 ppm / 1.0 ppm. Coupling ③ between the 3HB oxymethine proton and the 3HB methylene proton showed a cross peak at δ5.4 ppm / 2.6 ppm. Coupling ④ between the oxymethine protons of 3HB and the remaining methyl protons of 3HB was found at δ5.2 ppm / 1.3 ppm.

A 125-MHz ¹³ C-NMR spectrum of P (2HB-co-3HB) with chemical shift and broad spectrum of carbonyl carbon sites is shown in FIG.

The site (168.0-170.1 ppm) showed three groups of peaks with clear resolution, carbonyl carbon in the 2HB * -2HB sequence appeared at the peak of 168.7 ppm, 3HB * -3HB and 3HB-3HB * Carbonyl resonance in the sequence was found at 169.1 ppm. The 3HB * -2HB + 2HB-3HB * sequence was found at 169.5 ppm. The diad-sequence distribution of 2HB and 3HB was obtained from the peak area of carbonyl resonance, and the results are shown in Table 5.

2HB-co-3HB daid sequence data was compared with Bernoullian statistics applied to statistical random copolymerization.

Table 5

DSC and GPC analysis of P (50 mol% 2HB-co-50 mol% 3 HB) confirmed that amorphous P (2HB-co-3HB) was synthesized by recombinant E. coli, and P (2HB-co- The molecular weight of 3HB) was 20,000 (Mn) and 33,800 (Mw) with 1.69 PDI. Glass phase transition temperature was 9.7 degreeC.

As described above, the present invention has an effect of providing a polyhydroxyalkanoate containing 2-hydroxybutyrate, a biodegradable novel polymer as a monomer, and a recombinant microorganism capable of producing the same.

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims

A gene encoding a polyhydroxy alkanoate synthase and a gene encoding a propionyl-CoA transferase have been introduced, and a recombinant microorganism having a polyhydroxyalkanoate generating ability containing 2-hydroxybutyrate as a monomer. .
The microorganism according to claim 1, wherein the recombinant microorganism lacks a gene encoding lacdate dehydrogenase.
The method of claim 1, wherein the polyhydroxy alkanoate synthetase is Pseudomonas sp. PHA synthase or Pseudomonas sp. A recombinant microorganism, characterized in that it is a mutation enzyme of PHA synthase of 6-19.
The gene encoding the mutant of PHA synthase is selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 12. A recombinant microorganism having a base sequence corresponding to an amino acid sequence comprising one or more mutations.
The method of claim 3, wherein the gene encoding the mutant of PHA synthase is an amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in the amino acid sequence of SEQ ID NO: 12;

Amino acid sequence (C1310) in which E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 12; And

Recombinant microorganism, characterized in that in the amino acid sequence of SEQ ID NO: 12 E130D, S477F and Q481R has a base sequence corresponding to any one amino acid sequence selected from the group consisting of mutated amino acid sequence (C1312).
The method of claim 1, wherein the propionyl -CoA transferase is a recombinant microorganism, characterized in that the mutant enzyme of the transferase or propionyl propionyl -CoA -CoA transferase of C. propionicum of C. propionicum Pct540.
The gene encoding lacdate dehydrogenase is deleted, the gene encoding polyhydroxy alkanoate synthase and the gene encoding propionyl-CoA transferase, D-2 hydroxy acid dehydrogenase A gene encoding a gene, a gene encoding a simalate synthase, 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate / (R) -2-methylmalate dehydratase (leuBCD), and 2-hydroxybutyrate are monomers. Recombinant microorganisms having a polyhydroxyalkanoate-generating ability.
8. The method of claim 7, wherein said polyhydroxy alkanoate synthetase is Pseudomonas sp. PHA synthase or Pseudomonas sp. Recombinant microorganism, characterized in that PhaC1 which is a mutation of PHA synthase of 6-19.
The method of claim 7, wherein the propionyl -CoA transferase is a recombinant microorganism, characterized in that the mutant enzyme of the transferase or propionyl propionyl -CoA -CoA transferase of C. propionicum of C. propionicum Pct540.
8. The recombinant microorganism of claim 7, wherein the D-2 hydroxy acid dehydrogenase is derived from Lactobacillus lactis .
8. The recombinant microorganism of claim 7, wherein the simaleate synthase is derived from Methanococcus jannaschii .
The recombinant microorganism according to claim 7, wherein the 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate / (R) -2-methylmalate dehydratase (leuBCD) enzyme is derived from E. coli.
Culturing the recombinant microorganism of claim 1 in 2-hydroxybutyrate and 3-hydroxybutyrate containing media to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as monomer; And obtaining a polyhydroxyalkanoate containing the produced 2-hydroxybutyrate as a monomer. A method for producing a polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer.
Culturing the recombinant microorganism of claim 7 in a medium containing glucose and 3-hydroxybutyrate to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer; And obtaining a polyhydroxyalkanoate containing the produced 2-hydroxybutyrate as a monomer. A method for producing a polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer.
Culturing the recombinant microorganism of claim 7 in a glucose-containing medium to produce polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer; And obtaining a polyhydroxyalkanoate containing the produced 2-hydroxybutyrate as a monomer. A method for producing a polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer.
Polyhydroxyalkanoate containing 2-hydroxybutyrate as a monomer.