WO2018159965A1 - Method for producing polyhydroxyalkanoate using 2-hydroxyisocaproate-coa transferase - Google Patents

Abstract

The present invention relates to a recombinant microorganism to which a gene coding for 2-hydroxyisocaproate-CoA transferase and a gene coding for polyhydroxyalkanoate synthase are introduced and which has a potential of producing polyhydroxyalkanoate bearing an aromatic monomer or a long-chain 2-HA monomer and a method for producing polyhydroxyalkanoate bearing an aromatic monomer or a long-chain 2-HA monomer, using the recombinant microorganism. According to the present invention, a biodegradable polymer bearing an aromatic monomer or a long-chain 2-HA monomer can be produced.

Description

Method for preparing polyhydroxyalkanoate using 2-hydroxyisocaproate-CoA transferase

The present invention relates to a method for producing a polyhydroxyalkanoate containing an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) as a monomer, more particularly 2-hydroxyiso. A gene encoding a caproate-CoA transferase and a gene encoding a polyhydroxyalkanoate synthetase have been introduced, and have a polyhydroxyalkanoate generating ability containing an aromatic monomer or a long chain 2-HA as a monomer. A method for producing a polyhydroxyalkanoate containing a recombinant microorganism and an aromatic monomer or a long chain 2-HA using the recombinant microorganism as a monomer.

Polyhydroxyalkanoates (PHAs) are biological polyesters synthesized by a variety of microorganisms. These polymers are thermoplastics with biodegradable and biocompatible properties, and can have similar industrial properties as petroleum-based polymers, enabling a variety of industrial biomedical applications and resulting from renewable resources (Lee, SY Biotechnol. Bioeng . 49: 1 1996).

PHAs are classified into short-chain-length PHAs (SCL-PHAs) having a short carbon number and medium-chain-length PHAs (MCL-PHAs) having a long carbon number according to side chain lengths. Cloning of PHA synthetic genes derived from microorganisms such as Ralstonia eutropha, pseudomonas, Bacillus, etc. to produce recombinant microorganisms through which various PHAs were synthesized (Qi et al., FEMS Microbiol. Lett., 157: 155, 1997; Qi et al., FEMS Microbiol. Lett ., 167: 89, 1998; Langenbach et al., FEMS Microbiol. Lett., 150: 303, 1997; WO 01/55436; US 6,143,952; WO 98/54329; WO 99/61624 ).

Those with short side chains, such as PHB, a homopolymer of R-3-hydroxy butyric acid, are crystalline thermoplastics and have low-elastic properties that break well. On the other hand, MCL-PHA with long side chains has higher elasticity. PHB has been known for about 70 years (Lemoigne & Roukhelman, 1925). MCL-PHA, on the other hand, is relatively recently known (deSmet et al., J. Bacteriol . 154: 870-78 1983). These copolymers may be represented by PHB-co-HX, where X refers to 3 hydroxyalkanoate or alkanoate or an alkenoate of 6 to more carbons. Suitable examples of copolymers of certain two monomers include poly PHB-co-3-hydroxyhexanoate (Brandl et al., Int. J. Biol. Macromol . 11:49, 1989; A mos & McInerey, Arch Microbiol. , 155: 103, 1991; US 5,292,860).

Biosynthesis of PHA undergoes a process in which hydroxy acids are converted to hydroxyacyl-CoA by CoA-transferase or CoA-ligase, and the converted hydroxyacyl-CoA is polymerized by PHA synthase. For natural PHA synthase, the activity for 2-hydroxyacyl-CoA is much lower than for 3-hydroxyacyl-CoA. Recently, however, the inventors of Pseudomonas sp. 6-19 PHA synthase (PhaC1ps6-19) was genetically engineered to use lactyl-CoA, a type of 2-hydroxyacyl-CoA, as a substrate (WO08 / 062996; Yang et al., Biotechnol. Bioeng , 105: 150, 2010; Jung et al., Biotechnol. Bioeng ., 105: 161, 2010). PhaC1ps6-19 has a wide variety of substrate specificities and converts various types of 2-hydroxy acids to 2-hydroxyacyl-CoA because it can use lactyl-CoA, a type of 2-hydroxyacyl-CoA, as a substrate. Developing a feasible system would allow the synthesis of new PHAs containing different types of 2-hydroxy acids.

Accordingly, the present inventors have made intensive efforts to develop a method for biosynthesis of PHA containing a new 2-hydroxy acid. As a result, the enzyme converts 2-hydroxy acid using acetyl-CoA into 2-hydroxyacyl-CoA. When screening and using the enzyme, it was confirmed that it is possible to produce a variety of 2-hydroxyacyl-CoA under in vitro conditions, it was confirmed that it is possible to produce a variety of PHA by using this, to complete the present invention .

Summary of the Invention

An object of the present invention is to provide a recombinant microorganism having a polyhydroxyalkanoate producing ability containing an aromatic monomer or a long chain 2-HA as a monomer.

Another object of the present invention to provide a method for producing a polyhydroxyalkanoate containing an aromatic monomer or a long chain 2-HA as a monomer using the recombinant microorganism.

In order to achieve the above object, the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthase in a microorganism having acetyl-CoA generating ability from a carbon source. Provided is a recombinant microorganism having a polyhydroxyalkanoate-producing ability, which is introduced and contains an aromatic monomer or a long chain 2-HA as a monomer.

The present invention also comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing an aromatic monomer or a long chain 2-HA as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced aromatic monomer or long chain 2-HA as a monomer. Provided are methods for preparing canoate.

The present invention also relates to a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-de) in a microorganism having acetyl-CoA generating ability from a carbon source. Oxy-D-arabino-heptulsonate-7-phosphate) synthetase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate dehydrogenase A recombinant microorganism having a polyhydroxyalkanoate-producing ability containing a phenyl lactate as a monomer and amplified gene is provided.

The present invention also comprises the steps of (a) culturing the recombinant microorganism, to produce a polyhydroxyalkanoate containing phenyl lactate as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced phenyllactate as a monomer. It provides a method for producing polyhydroxyalkanoate containing a phenyllactate as a monomer.

The present invention also relates to a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-de) in a microorganism having acetyl-CoA generating ability from a carbon source. Oxy-D-arabino-heptulsonate-7-phosphate) synthetase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate dehydrogenase A gene encoding a hydroxymandelate synthase, a gene encoding a hydroxymandelate oxidase, and a gene encoding a D-mandelate dehydrogenase, and containing a mandelate as a monomer. Provided is a recombinant microorganism having hydroxyalkanoate producing ability.

The present invention also comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing mandelate as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced mandelate as a monomer, thereby providing a method for preparing polyhydroxyalkanoate containing a mandelate as a monomer.

Figure 1 shows the biosynthetic metabolic pathway of the aromatic polyester according to the present invention, A is the metabolic pathway when using FldA (cinnamoyl-CoA: phenyllactate CoA-transferase), B is HadA (2- Metabolic pathways using hydroxyisocaproate-CoA transferase).

Figure 2 shows the result of comparing the amino acid sequence homology of HadA and FldA.

3 shows the result of purifying HadA using His-tag, and b shows the result confirming whether HadA can use acetyl-CoA as a CoA donor.

Figure 4 shows that HadA uses acetyl-CoA as CoA donor, mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy The conversion of oxy-3-phenylpropionate and 4-hydroxy benzoic acid to the corresponding CoA derivatives was confirmed by LC-MS analysis after in vitro enzyme assay.

5 is a molecular formula of the CoA conversion reaction of various substrates that HadA can convert.

Figure 6 shows the results of increasing the production of D-phenyl lactate by performing metabolic analysis according to in silico genome scale metabolic flux analysis.

Figure 7 shows the results of the analysis of poly (3HB-co-D-phenyl lactate) produced by E. coli XB201TBAL.

Figure 8 Poly (3HB-co-D-phenyllactate-co-3-hydroxy-3-phenylpropionate) and poly (3HB-co-D-phenyllactate-co-D produced by Escherichia coli XB201TBAL -Mandelate) shows the result of analysis.

9 shows the results of poly (3HB-co-D-phenyllactate) production in E. coli XB201TBAL strains expressing PhaAB under five promoters of different intensities.

10A and 10B show the production of poly (3HB-co-D-phenyllactate) by fed-batch fermentation of E. coli XB201TBAL strain expressing AroGfbr, PheAfbr, FldH, HadA and PhaC1437 in MR medium containing 3HB. C and d are fed by a fed-batch fermentation of the E. coli XB201TBAL strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters without addition of 3HB to obtain poly (3HB-co-D-phenyllactate). Production results are shown, and e and f are fed by a fed-batch fermentation of the E. coli XB201TBAF strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters without addition of 3HB, and thus poly (3HB-co-D-phenyllac Tate) is produced.

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.

Aromatic polyesters are essential plastics produced mainly from petroleum. In the present invention, an aromatic polyester or a long chain 2-hydroxyalkanoate is prepared from glucose by metabolic engineering E. coli expressing polyhydroxyalkanoate (PHA) synthase and coenzyme A (CoA) transferase. The method of producing the monomer polymer by one-step was established.

In one embodiment of the present invention, cinnamoyl-CoA: phenyllactate CoA-transferase (FldA) and 4-Cuma have been identified by in vitro assays to produce PHAs containing phenyllactate as aromatic polyesters. Rate: CoA ligase (4CL) was expressed in D-phenyllactate producing E. coli strains along with PHA synthase. The strain was prepared using poly (16.8 mol% D-lactate-co-80.8 mol% 3HB-co-1.6mol% D-phenyllactate-co-0.8mol using Cinnamoyl-CoA generated in vivo as a CoA donor. % D-4-hydroxyphenyllactate) polymer was produced at a content of 12.8% by weight of dry cell weight.

However, since the range of aromatic substrate use of phenyllactate CoA-transferase (FldA) is very narrow, 2-isocaprenoyl-CoA capable of producing various aromatic hydroxyacyl CoAs using acetyl-CoA as CoA donor: 2-hydroxyisocaproate CoA-transferase (HadA) was identified and selected for use in aromatic PHA production. In order to mass produce aromatic PHAs containing high mole fractions of D-phenyllactate, an optimal metabolic pathway was first designed to overproduce the monomer D-phenyllactate.

In one aspect of the present invention, in order to prepare an E. coli engineered to have an optimal metabolic pathway for D-phenyllactate production, over-expression of feedback resistance aroG, pheA and fldH genes in tyrR deficient E. coli, and competitive metabolism The pathways (pflB, poxB, adhE and frdB) were deleted and the tyrB and aspC genes were further deleted following in silico genome scale metabolic flux analysis. The metabolically engineered Escherichia coli produced 1.62 g / L of D-phenyllactate. When HadA and PHA synthase were expressed in the D-phenyllactate over-producing strain, 15.8% by weight of poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate) of dry cell weight was produced. Confirmed. In addition, by making polyesters comprising 4-hydroxyphenyllactate, mandelate and 3-hydroxy-3-phenylpropionate, the possibility of producing various aromatic polyesters was confirmed.

Therefore, in one aspect, the present invention, in a microorganism having the ability to produce acetyl-CoA from a carbon source, a gene encoding 2-hydroxyisocaproate-CoA transferase and a gene encoding a polyhydroxyalkanoate synthase is introduced The present invention relates to a recombinant microorganism having a polyhydroxyalkanoate generating ability containing an aromatic monomer or a long chain 2-HA as a monomer.

In the present invention, the long chain 2-HA means 2-hydroxyalkanoate having 6 to 8 carbon atoms.

In the present invention, the aromatic monomer or long-chain 2-HA monomer is 2-hydroxyisocaproate, 2-hydroxyhexanoate, 2-hydroxyoctanoate, phenyl lactate, 2-hydroxy-4 It may be characterized in that it is selected from the group consisting of -phenylbutyrate, 3-hydroxy-3-phenylpropionate, 4-hydroxybenzoic acid and mandelate.

In the present invention, the polyhydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:

An amino acid sequence comprising one or more variants 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: 2;

Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).

In the present invention, the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630.

In the present invention, the 2-hydroxyisocaproate-CoA transferase may be characterized by using acetyl-CoA as CoA donor.

The microorganism of the present invention is a gene encoding β-ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.

In another aspect, the present invention comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing an aromatic monomer or long chain 2-HA as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced aromatic monomer or long chain 2-HA as a monomer. It relates to a method for producing a canoate.

In one embodiment of the present invention, whether Pct (Propionyl-CoA transferase) used for polyhydroxyalkanoate synthesis can activate phenyllactate and mandelate with phenyllactyl-CoA and mandelyl-CoA, respectively. . Mutation of Pctcp (Pct540) has been successful in in vivo production of polyesters comprising 2-hydroxy acids such as glycolic acid, lactic acid, 2-hydroxybutyl acid, 2-hydroxyisovalorate and various hydroxy acids. Since it is used, Pct540 can be said to have a broad substrate spectrum with respect to carbon number and hydroxyl position. However, Pct540 was found to have no catalytic activity for phenyllactate and mandelate, and therefore, in the present invention, for the production of aromatic copolymers, a novel CoA-transition capable of activating CoA derivatives corresponding to aromatic compounds We wanted to find an enzyme.

Cinnamoyl-CoA: Phenyllactate CoA-transferase (FldA) of Clostridium sporogenes has been reported to convert phenyllactate to phenyllactyl-CoA using cinnamoyl-CoA as a CoA donor (Dickert, S. et al., Eur. J. Biochem . 267: 3874, 2000). Cinnamoyl-CoA is a non-natural metabolite of Escherichia coli. Therefore, to confirm the use of acetyl-CoA, a rich metabolite in cells, as a CoA donor, Clostridium botulinum A str. FldA from ATCC 3502 was tested. However, C. botulinum A str. It was confirmed that FldA of ATCC 3502 does not have the catalytic activity of generating phenyllactyl-CoA using acetyl-CoA as CoA donor.

On the other hand, it is known that Streptomyces coelicolor 4-coumarate: CoA ligase (4CL) plays an important role in phenylpropanoid metabolism, which produces precursors of plant secondary metabolites such as lignin, flavonoids and phytoalexin (Kaneko, M. et al, J. Bacteriol. 185: 20, 2003). Therefore, in one aspect of the present invention, a biosynthetic pathway for synthesizing cinnamoyl-CoA from cinnamate was designed by introducing 4CL. The 4CL variant was used to convert cinnamates to cinnamoyl-CoA and cinnamoyl-CoA was used as CoA donor of FldA for phenyllactyl-CoA formation. As a result, phenyllactyl-CoA was successfully synthesized by sequential reaction of 4CL and FldA in vitro . These results confirmed that 4CL and FldA could be used for the production of phenyllactyl-CoA and that it could be used for producing aromatic polyesters. Similarly, it was confirmed that 4-hydroxyphenyllactate, another promising aromatic monomer, was also converted to 4-hydroxyphenyllactyl-CoA by in vitro sequential reaction of 4CL variant with FldA.

In the preparation of non-natural polyesters, it is important to select a variant of PHA synthase for polymerization of the corresponding CoA substrate. Therefore, Pseudomonas sp. MBEL 6-19 PHA synthase (PhaCPs6-19) variants were expressed in Escherichia coli XL1-Blue overexpressing AroGfbr, PAL, 4CL, FldA and Pct540. The prepared recombinant strain was cultured in MR medium to which 20 g / L glucose, 1 g / L D-phenyllactate and 1 g / L sodium 3-hydroxybutyrate (3HB) were added. Sodium 3-hydroxybutyrate (3HB) was converted by Pct540 to 3HB-CoA, which is the preferred substrate of PhaC, and was added to enhance the production of the polymer because it allows the production of sufficient amounts of PHA. E. coli XL1-Blue, which expresses different PHA synthase variants, can produce varying amounts of poly (D-lactate-co-3HB-co-D-phenyllactate) with different monomer compositions.

As a result of this experiment, PhaC1437 containing four amino acid substitutions (E130D, S325T, S477G and Q481K) in the PhaC variant was poly (18.3 mol% D-lactate-co-76.9 mol% 3HB-co-4.8 mol% D-phenyl Lactate) was produced at 7.8% by weight of the dry cell weight to identify the most suitable PhaC variant.

Next, E. coli was manipulated to generate D- phenyllactate from glucose in vivo . Biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulsonate-7-phosphate (DAHP), which is combined with phosphoenolpyruvate (PEP) by DAHP synthase. It is produced by the condensation of ellithrose-4-phosphate (E4P). The resulting DAHP is converted to phenylpyruvate (PPA) followed by D-lactate dihydrogenase (FldH) to D-phenyllactate (FIG. 1). Metabolic pathways for aromatic compound biosynthesis are known to be complexly controlled by various inhibitory mechanisms. Expression of DAHP synthase encoded by aroG and corismate mutase / prephenate dehydrogenase encoded by pheA is inhibited by L-phenylalanine (ribe, DE et al., J. Bacteriol. 127: 1085, 1976).

In the present invention, feedback inhibition resistance mutations AroGfbr [AroG (D146N)] and PheAfbr [PheA (T326P)] were constructed to release feedback inhibition by L-phenylalanine (Zhou, HY et al., Bioresour. Technol. 101: 4151, 2010; Kikuchi, Y. et al., Appl. Environ.Microbiol. 63: 761, 1997). AroGfbr, PheAfbr and C. botulinum A str. Escherichia coli XL1-Blue expressing FldH of ATCC 3502 produced 0.372 g / L of D-phenyllactate from 15.2 g / L of glucose. Overexpression of PAL, 4CL, FldA, Pct540 and PhaC1437 of the strain was poly (16.8 mol% D-lactate-co-80.8 mol% 3HB-co-1.6 mol% D-phenyllactate-co-0.8 mol% D -4-hydroxyphenyllactate) was increased to 12.8% by weight of the dry cell weight. There are two problems in preparing aromatic PHA containing D-phenyllactate. Firstly, the efficiency of polymer synthesis and the content of aromatic monomers are very low, which is due to the fact that FldA using Cinnamoyl-CoA as a CoA donor. It was thought to be due to inefficiency. The second is that the monomer spectrum of aromatic PHAs is very narrow. In vitro enzyme analysis showed that FldA can transfer CoA to phenyllactate and 4-hydroxyphenyllactate, but mandelate, 2-hydroxy-4-phenylbutylate, 3-hydroxy-3-phenylpropio It was confirmed that it could not be transferred to substrates such as nate and 4-hydroxy benzoic acid.

In the present invention, in order to solve the above problem, an enzyme having a broad spectrum of aromatic substrate was found using acetyl-CoA as CoA donor. An array similarity analysis was performed to identify homologous enzymes for FldA and 2-isocaprenoyl-CoA: 2-hydroxyisocapronate of Clostridium difficile having at least 48% amino acid sequence identity with FldA among various FldAs of different origins. CoA-transferase (HadA) was screened (FIG. 2). Since HadA is an enzyme known to convert CoA into 2-hydroxyisocaproate using isocaprenoyl-CoA as a CoA donor, the present invention investigated whether HadA can use acetyl-CoA as a CoA donor ( 3). In vitro enzyme analysis showed that HadA was able to activate phenyllactate as phenyllactyl-CoA using acetyl-CoA as a CoA donor (FIG. 4).

HadA also contains mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy-3-phenylpropionate and 4 It was confirmed through LC-MS analysis after in vitro assay that hydroxy benzoic acid can be converted into the corresponding CoA derivative (FIGS. 4 and 5). Therefore, HadA has the potential to produce various aromatic polyesters more efficiently by using acetyl-CoA as a CoA donor.

Next, E. coli XL1-Blue strains expressing AroGfbr, PheAfbr and FldH, which produce a small amount of D-phenyllactate (0.372 g / L) from glucose, were used to increase the amount of aromatic monomer produced by metabolic engineering. The yield was raised through the operation. E. coli XBT strains expressing AroGfbr, PheAfbr and FldH, which deleted TyrR, a dual transcriptional regulator that regulates aromatic amino acid biosynthesis, were prepared, and the E. coli XBT strain was 0.5 g / L D from 16.4 g / L glucose. -Phenyllactate produced 30% higher productivity than the E. coli XL1-Blue strain without TyrR deletion. To eliminate a path conflict with D-phenyllactate biosynthesis, in E. coli XBT, poxB (a gene encoding pyruvate oxidase), a pyruvate oxidase), pflB (a gene encoding pyruvate formate lyase), Escherichia coli XB201T was constructed by deleting adhE (gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase) and frdB (gene encoding fumarate reductase). E. coli XB201T strains expressing AroGfbr, PheAfbr and FldH produced 0.55 g / L of D-phenyllactate from 15.7 g / L of glucose, indicating a 10% higher yield than E. coli XBT.

In addition, metabolic analysis according to in silico genome scale metabolic flux analysis was performed to further increase D-phenyllactate production (FIG. 6). The tyrB gene encoding tyrosine aminotransferase and the aspC gene encoding aspartic acid aminotransferase were removed from the E. coli XB201T strain, reducing L-phenylalanine biosynthesis to enhance carbon flow to D-phenyllactate. The resulting E. coli XB201TBA strain expressing AroGfbr, PheAfbr and FldH produced 1.62 g / L of D-phenyllactate from 18.5 g / L of glucose, which greatly increased the yield of E. coli. It is 4.35 times higher than D-phenyllactate production of XL1 Blue strain.

Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was incubated in a medium containing 20 g / L glucose and 1 g / L sodium 3HB to obtain poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate). Was produced at a content of 15.8% by weight of dry cell weight (FIG. 7).

Therefore, in another aspect, the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, in a microorganism having acetyl-CoA generating ability from a carbon source, Gene encoding DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate di The present invention relates to a recombinant microorganism having a polyhydroxyalkanoate-producing ability, in which a gene encoding a hydrogenase has been introduced and containing phenyllactate as a monomer.

In the present invention, the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630, the hydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp . It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:

An amino acid sequence comprising one or more variants 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: 2;

Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).

In the present invention, the gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 8 The gene encoding the corismate mutase / prephenate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 9, wherein the D-lactate dehydrogeze The gene encoding the naze may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.

In the present invention, the gene encoding the introduced D-lactate dehydrogenase may be characterized as being a fldH gene that replaces the ldhA gene.

The microorganism of the present invention is a gene encoding β-ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.

When the expression level of the gene (phaA) encoding β-ketothiolase and gene (phaB) encoding acetoacetyl-CoA reductase introduced in the present invention is controlled through the strength of the promoter, The mole fraction of D-phenyllactate monomer can be controlled.

In one embodiment of the present invention, five different plasmids expressing PhaAB under five promoters of different intensities were constructed and introduced into XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA, and D- with decreasing PhaAB expression. The molar fraction of phenyllactate monomer was found to increase (Figures 9a, b and Table 7). These results suggest that by controlling the metabolic flux, aromatic polyesters with various aromatic monomer mole fractions can be produced.

In the present invention, the recombinant microorganism is a tyrR gene, a gene encoding pyruvate oxidase, a gene encoding pyruvate formate lyase, a gene encoding acetaldehyde dehydrogenase, a gene encoding fumarate reductase, tyrosine amino One or more genes selected from the group consisting of a gene encoding the transferase and a gene encoding the aspartic acid aminotransferase may be deleted.

In another aspect, (a) culturing the recombinant microorganism, to produce a polyhydroxyalkanoate containing phenyl lactate as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced phenyllactate as a monomer. The present invention relates to a method for preparing polyhydroxyalkanoate containing a phenyllactate as a monomer.

In order to confirm that the method can be used for the preparation of various aromatic polymers, mandelate was tested using monomers. This is because polymandelate, the single polymer of mandelate, is a pyrolysis resistant polymer having a relatively high Tg of 100 ° C., while the material properties are similar to polystyrene. Polymandelate is chemically synthesized by ring-opening polymerization of cyclic dimer of mandelate produced in the petroleum industry. Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was cultured in a medium containing 1 g / L sodium 3HB and 0.5 g / L D-mandelate to obtain poly (55.2 mol% 3HB-co-43 mol% D-phenyl Lactate-co-1.8 mol% D-mandelate) was prepared at a 11.6 wt% content of dry cell weight (FIGS. 8 a, b). In the present invention, an aromatic copolymer including D-mandelate was successfully prepared using D-mandelate as a substrate, and then, to produce D-mandelate in vivo by metabolic engineering. Hydrolysis of Amycolatopsis orientalis-derived hydroxymandelate synthetase (HmaS), S. coelicolor, in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA to produce an aromatic copolymer comprising D-mandelate from glucose Roxymandelate oxidase (Hmo) and D-mandelate dehydrogenase (Dmd) from Rhodotorula graminis were expressed. When the engineered strains were cultured in a medium containing 20 g / L glucose and 1 g / L sodium 3HB, 16.4% by weight of poly (92.9 mol% 3HB-co-6.3 mol% D-phenyl) Lactate-co-0.8 mol% D-mandelate).

Therefore, in another aspect, the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, in a microorganism having acetyl-CoA generating ability from a carbon source, Gene encoding DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate di Genes encoding hydrogenase, genes encoding hydroxymandelate synthase, genes encoding hydroxymandelate oxidase and genes encoding D-mandelate dehydrogenase have been introduced. The present invention relates to a recombinant microorganism having a polyhydroxyalkanoate producing ability contained as a monomer.

In the present invention, the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630, the hydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp . It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:

An amino acid sequence comprising one or more variants 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: 2;

Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).

In the present invention, the gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 8 The gene encoding the corismate mutase / prephenate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 9, wherein the D-lactate dehydrogeze The gene encoding the naze may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.

In the present invention, the gene encoding the hydroxymandelate synthetase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 11, the gene encoding the hydroxymandelate oxidase May be a gene encoding an amino acid sequence represented by SEQ ID NO: 12, and the gene encoding the D-mandelate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 13 Can be.

The microorganism of the present invention is a gene encoding β-ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.

In another aspect, the present invention provides a method for producing a polyhydroxyalkanoate containing mandelate as a monomer by culturing the recombinant microorganism; And (b) obtaining a polyhydroxyalkanoate containing the produced mandelate as a monomer.

In addition, in the present invention, in order to determine whether the production of polyhydroxyalkanoate containing various long-chain 2-HA is possible using the recombinant strain of the present invention, 2-hydroxyisocapro which is a long-chain 2-HA monomer 8 (2HIC), 2-hydroxyhexanoate (2HH) and 2-hydroxyoctanoate (2HO) were used as monomers to confirm polymer production capacity. As a result, 2-hydroxyisocaproate, It was confirmed that a copolymer containing 2-hydroxyhexanoate or 2-hydroxyoctanoate was produced, and as the concentration of 2-HA contained in the medium increased, the mole fraction of the monomer contained in the copolymer increased. Was confirmed (Table 4, Table 5 and Table 6).

Accordingly, the present invention provides a recombinant microorganism having the ability to produce polyhydroxyalkanoate containing the aromatic monomer or the long-chain 2-HA as a monomer, in a further aspect, 2-hydroxyisocaproate, 2-hydroxy. Culturing in a medium containing a compound selected from the group consisting of oxyhexanoate and 2-hydroxyoctanoate; And (b) obtaining a polyhydroxyalkanoate containing as a monomer a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate Regarding a method for producing polyhydroxyalkanoate containing as a monomer a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate will be.

In the present invention, aromatic polymer production was confirmed using 3-hydroxy-3-phenylpropionate (3HPh) as another aromatic monomer capable of generating PHA. Escherichia coli XB201TBA strains were cultured in a medium containing 20 g / L glucose, 0.5 g / L 3-hydroxy-3-phenylpropionic acid, and 1 g / L sodium 3HB to obtain a poly (33.3 mol% 3HB-co-18 mol% D-phenyl Lactate-co-48.7 mol% 3HPh) was confirmed to produce 14.7% by weight of the dry cell weight (Fig. 8c, d). These results suggest that the HadA and mutated PHA synthase developed in the present invention can be widely used in the preparation of various aromatic polyesters.

Finally, the material properties of aromatic PHA produced by metabolically engineered Escherichia coli were investigated. Poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate) was amorphous, and the Tg increased significantly to 23.86 ° C. despite decreasing molecular weight as the mole fraction of D-phenyllactate in the copolymer increased It was. In addition, copolymers containing aromatics in the polymers reduced crystallinity. It is assumed that the aromatic ring of the polymer interferes with the crystallization of P (3HB). In the case of P (3HB), the resultant copolymer exhibited high brittleness due to the strong crystallinity, whereas in the resulted copolymer, the crystallinity was lowered and the Tg was improved to improve the mechanical toughness.

In the present invention, a bacterial platform system has been developed for the production of various aromatic polyesters. The aromatic polymer production system of the present invention identifies CoA-transferases having a new broad substrate range for activating aromatic compounds into their CoA derivatives, and in vivo, PHA synthase variants capable of polymerizing their aromatic CoA derivatives. The pathway to overproduce aromatic monomers was established through the design and optimization of metabolism.

Using several aromatic monomers in various embodiments of the present invention, as demonstrated, such systems can be used for the preparation of various aromatic polymers. For example, according to the present invention, HadA (or related enzymes) and PHA synthetase can be engineered to accommodate the desired aromatic monomer. The bacterial platform system developed in the present invention can contribute to establishing a bioprocess for the production of aromatic polyesters from renewable non-food biomass.

 In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. It includes. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli, strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli, such as Bacillus subtilis, Salmonella typhimurium or Serratia marghesen Still other enterobacteria such as marcescens and various Pseudomonas strains can be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen). When the cloning is performed using the pET family vector, histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified. In order to separate the expressed protein from the cloned gene, a general method known in the art may be used, and specifically, it may be separated by a chromatographic method using Ni-NTA His-binding resin (Novagen). . In the present invention, the recombinant vector may be characterized in that the pET-SLTI66, the host cell may be characterized in that E. coli or Agrobacterium.

The expression “expression control sequence” in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from cytomegalovirus, and the like are preferably used. To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof. The T7 promoter can be usefully used to express the proteins of the invention in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once the expression vector is in the host cell, it can replicate independently of the host chromosomal DNA and several copies of the vector and the inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture. As a screening method when cloning the cDNA of a protein according to the present invention by expression cloning, a binding method (binding method), a panning method, a film emulsion method and the like can be applied.

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.

In the following examples, E. coli was used as a recombinant microorganism, but any microorganism that produces acetyl-CoA from a carbon source can be used without limitation, and includes genus Alcaligenes, Pseudomonas, Escherichia, Ralstonia genus, Bacillus genus, Corynebacterium and the like can be used.

Recombinant strains, plasmids and primers used or produced in the present invention are shown in Tables 1-3.

1 Datsenko, K. A. & Wanner, B. P Natl Acad Sci USA 97: 6640, 2000.

2 Lee, KH et al., Molecular Systems Biology 3, doi: ARTN 149 10.1038 / msb4100196, 2007.

3 Palmeros, B. et al. Gene 247: 255, 2000.

4 Park, SJ et al., Metab Eng 20, 20, 2013.

5 Yang, TH et al. Biotechnol Bioeng 105: 150, 2010.

6 Yang, TH et al., Appl Microbiol Biotechnol 90: 603, 2011.

7 Choi, SY et al., Nat Biotechnol 34: 435, 2016.

8 Knobloch, K. H. & Hahlbrock, K., Archives of Biochemistry and Biophysics 184: 237, 1977.

9 Kaneko, M. et al., J Bacteriol 185: 20, 2003.

Example 1 Construction of Recombinant 2-hydroxyisocapronate CoA-Transferase

Acetyl-CoA was used as CoA donor to find enzymes with broad spectrum of aromatic substrates. An array similarity assay was performed to identify homologous enzymes for FldA and 2-isocaprenoyl-CoA: 2-hydroxyisocaproate of Clostridium difficile having at least 48% amino acid sequence identity with FldA among various FldAs of different origins. CoA-transferase (HadA, SEQ ID NO: 1) was screened (FIG. 2).

In order to prepare a recombinant vector containing a gene encoding HadA, the chromosomal DNA of Clostridium diffsile 630 strain was used as a template, and PCR was performed using HadA-hisF and HadA-hisR primers to obtain a C terminus. A his_HadA gene segment encoding 2-hydroxyisocaproate-CoA transferase with his-tag was constructed.

Next, the prepared his_HadA fragment was treated with restriction enzymes (NdeI and NotI) to the pET22b plasmid undergoing strong gene expression of the T7 promoter, followed by T4 DNA ligase, and his_HadA fragment digested with restriction enzyme and pET22b plasmid was conjugated to prepare a recombinant plasmid pET22b_hisHadA (FIG. 2).

The pET22b_hisHadA was introduced into Escherichia coli XL1-Blue (Stratagene Cloning Systems, USA), cultured, and IPTG was added to induce HadA expression, followed by Ni-NTA spin kit (Quiagen, Germany) using His-tag. HadA was purified from the culture medium at (Fig. 3a).

Example 2: Determination of Substrate Diversity of 2-hydroxyisocaproate CoA-Transferase

In order to confirm that HadA can use acetyl-CoA as a donor, an in vitro assay was performed using HadA prepared in Example 1.

10 μg of HadA was added to 50 mM phosphate buffer (pH 7.5) containing 0.1 mM acetyl-CoA and 10 mM substrate and the reaction was performed at 30 ° C. for 10 minutes. After the reaction, 0.1 mM oxal acetic acid, 5 μg citrate synthase and 0.5 mM 5.5'-dithiobis- (2-nitrobenzoic acid) (DTNB) were added. The amount of free CoA was then analyzed by measuring the absorbance at 412 nm (FIG. 3B).

Analysis of the resulting aliphatic and aromatic acyl-CoA was performed on LC-MS (Agilent 1100 series and LC / MSD VL, Agilent) equipped with Eclipse XDB-C18 column (5 μm, 4.6 × 150 mm, Agilent).

As a result, as shown in FIG. 4, HadA was treated with acetyl-CoA as CoA donor, mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4 Using -phenylbutyrate, 3-hydroxy-3-phenylpropionate and 4-hydroxy benzoic acid as substrates, it was confirmed that the substrate can be converted to the corresponding CoA derivative.

Figure 5 shows the molecular formula of the CoA conversion reaction of the various substrates that HadA can convert.

Example 3: Construction of Recombinant Strains with Increased Aromatic Monomer Production

E. coli was engineered to produce D-phenyllactate from glucose in vivo. Biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulsonate-7-phosphate (DAHP), which is combined with phosphoenolpyruvate (PEP) by DAHP synthase. It is produced by the condensation of erythrose-4-phosphate (E4P). The resulting DAHP is converted to phenylpyruvate (PPA) followed by D-lactate dihydrogenase (FldH) to D-phenyllactate (FIG. 1). Metabolic pathways for aromatic compound biosynthesis are known to be complexly controlled by various inhibitory mechanisms. Expression of DAHP synthase encoded by aroG and corismate mutase / prephenate dehydrogenase encoded by pheA is inhibited by L-phenylalanine (ribe, DE et al., J. Bacteriol. 127: 1085, 1976).

In the present invention, the feedback inhibition resistance mutants AroGfbr [AroG (D146N)] and PheAfbr [PheA (T326P)] were constructed to release feedback inhibition by L-phenylalanine (Zhou, HY et al., Bioresour. Technol . 101: 4151, 2010; Kikuchi, Y. et al., Appl. Environ.Microbiol . 63: 761, 1997). AroGfbr, PheAfbr and C. botulinum A str. E. coli XL1-Blue expressing the FldH of ATCC 3502 was constructed.

3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene (aroG), a feedback inhibition resistance mutation, was constructed using primers AroG-F and AroG-R to construct pKM212-AroGfbr. PCR product was prepared using the plasmid pTyr-a (Na, D. et al., Nature Biotechnol. 31: 170, 2013) as a template, and the PCR product was purified using pKM212 using restriction enzymes (EcoRI / HidIII). PKM212-AroGfbr was constructed in conjunction with -MCS (Park, SJ et al., Metab. Eng . 20:20, 2013).

The pKM212-AroGfbrPheAfbr plasmid was constructed as follows. First, 991 bp DNA fragments from genomic DNA of Escherichia coli were amplified by PCR using primers PheA-F and PheAmut-R of a single mutated base (T976G). Second, a 200 bp DNA fragment was amplified from genomic DNA of Escherichia coli using a single mutated base (A976C) and PheAmut-F primer of PheA-R.

Then, using the mixed two fragments as a template, a DNA fragment of 1161bp was amplified by the primers PheA-F and PheA-R by overlap PCR. The PCR product was linked to pKM212-AroGfbr prepared above using restriction enzyme (HindIII).

To construct pACYC-FldH, C. botulinum A str. D-lactate dehydrogenase (fldH) of ATCC 3502 was used. Codon usage frequency of the fldH gene was carried out using E. coli-optimized E. coli codon-optimized fldH gene using primers FldH-F and FldH-R and pUC57-FldHopt (GenScript, Piscataway, NJ, USA) as a template.

 The PCR product was linked with pTrc99A (Pharmacia, Biotech, Sweden) using restriction enzymes (BamHI / HindIII) to prepare pTrc-FldH. Next, the fldH gene coupled to the trc promoter and rrnB terminator was amplified by PCR using primers Trc-F and Ter-R using pTrc-FldH as a template. The amplified PCR product was linked with pACYC184KS (Korean Patent Publication No. 2015-0142304) using restriction enzymes (XhoI / SacI) to obtain pACYC-FldH.

PKM212-AroGfbrPheAfbr and pACYC-FldH prepared above were introduced into E. coli XL1-Blue to prepare recombinant E. coli expressing AroGfbr, PheAfbr and FldH.

The Escherichia coli produced 0.372 g / L of D-phenyllactate when incubated in MR medium containing 15.2 g / L of glucose.

The MR medium comprises 6.67 g KH 2 PO 4, 4 g (NH 4) 2 HPO 4, 0.8 g MgSO 4 .7H 2 O, 0.8 g citrate and 5 ml of trace metal solution, the trace metal solution being 0.5 M HCl: 10 g FeSO 4 · 7H 2 O, 2g CaCl 2, 2.2g ZnSO 4 .7H 2 O, 0.5g MnSO 4 .4H 2 O, 1g CuSO 4 .5H 2 O, 0.1g (NH 4) 6 Mo 7 O 24 .4H 2 O and 0.02 g Na 2 B 4 O 7 .10H 2 O.

In order to increase the amount of aromatic monomer produced by metabolic engineering, E. coli XL1-Blue strains expressing the AroGfbr, PheAfbr and FldH producing small amounts of D-phenyllactate (0.372 g / L) from glucose were subjected to metabolic engineering. Yield was increased through. E. coli XBT strains expressing AroGfbr, PheAfbr and FldH, which deleted TyrR, a dual transcription regulator that regulates aromatic amino acid biosynthesis, were prepared.

Deletion of the tyrR gene in E. coli expressing AroGfbr, PheAfbr and FldH was performed using a one step inactivation method (Datsenko, KA et al., Proc. Natl Acad. Sci. USA 97: 6640, 2000).

The E. coli XBT strain was cultured in MR medium containing 16.4 g / L glucose and produced 0.5 g / L D-phenyllactate, which was 30% higher than the E. coli XL1-Blue strain without tyrR. Productivity was shown.

To eliminate pathways that conflict with D-phenyllactate biosynthesis, poxB (a gene encoding pyruvate oxidase), pflB (a gene encoding pyruvate formate lyase), adhE (acetaldehyde dehydrogenase / alcohol dehydrogenase) in E. coli XBT E. coli XB201T was deleted, which deleted the enzyme encoding the gene) and frdB (the gene encoding fumarate reductase).

E. coli XB201T strain produced 0.55 g / L of D-phenyllactate from 15.7 g / L of glucose, indicating a 10% higher yield than E. coli XBT.

In addition, metabolic analysis was performed according to in silico genome scale metabolic flux analysis to further increase D-phenyllactate production.

For in silico flux response analysis, the E. coli iJO1366 genome scale model consisting of 2251 metabolites and 1135 metabolites was used, and the effects of central and aromatic amino acid biosynthesis reactions on D-phenyllactate production were investigated. To reflect the XB201T strain of the present invention, gene knockout was reflected in the model by further adding the heterologous metabolic response (fldH gene) of D-phenyllactate biosynthesis to the model and setting the flux to zero. The rate of D-phenyllactate production was maximized as the objective function, while the central and aromatic amino acid biosynthetic reaction flux values slowly increased from minimum to maximum. The glucose response rate during the simulation was set to 10 mmol per 1 g of dry cell weight per hour. All simulations were run in a Python environment using the Gurobi Optimizer 6.0 and GurobiPy packages (Gurobi Optimization, Inc. Houston, TX). COBRApy32 was used to read, write, and work with COBRA-compliant SBML files.

As a result, as shown in FIG. 6, the tyrB gene encoding tyrosine aminotransferase and the aspC gene encoding aspartic acid aminotransferase were removed from the E. coli XB201T strain, and L-phenylalanine biosynthesis was reduced to D-phenyllactate. The carbon flow to the furnace was enhanced.

The E. coli XB201TBA strain produced as a result of the in silico flux response analysis produced 1.62 g / L of D-phenyllactate from 18.5 g / L of glucose, which greatly increased the yield of E. coli expressing AroGfbr, PheAfbr and FldH. It is 4.35 times higher than D-phenyllactate production of XL1-Blue strain.

Example 4 Preparation of Polyhydroxyalkanoate Containing Aromatic Monomer Using Recombinant Strains

In order to prevent D-lactate formation in XB201TBA, an additional deletion of the ldhA gene was made to produce the XB201TBAL strain. To prepare polyhydroxyalkanoates containing aromatic monomers, PhaC1437 and HadA were expressed in E. coli XB201TBAL strain.

In order to prepare a recombinant vector containing genes encoding PhaC1437 and HadA, chromosomal DNA of Clostridium difficile 630 strain was used as a template, and PCR was performed using HadA-sbF and HadA-ndR primers to prepare a recombinant vector. A hadA gene segment encoding isocaproate-CoA transferase was constructed. The amplified PCR product was linked with p619C1437-pct540 (Yang, TH et al. Biotechnol Bioeng 105: 150, 2010) using restriction enzymes (SbfI / NdeI) to obtain p619C1437-HadA. P619C1437-HadA prepared above was introduced into E. coli XB201TBAL to prepare recombinant E. coli expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA. The E. coli was cultured in MR medium containing 20 g / L glucose and 1 g / L sodium 3HB to convert poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate) to a content of 15.8% by weight of the dry cell weight. Produced (FIG. 7). Fed-batch also produced poly (52.3 mol% 3HB-co-47.7 mol% D-phenyllactate) at a 24.3 wt% content of dry cell weight (FIGS. 10a, b).

Example 5 Preparation of Polyhydroxyalkanoates Containing Various Aromatic Monomers Using Recombinant Strains

In order to confirm that the system using E. coli XB201TBAL can be used for the preparation of various aromatic copolymers, mandelate was tested as a monomer.

Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was incubated in MR medium containing 1 g / L sodium 3HB and 0.5 g / L D-mandelate, resulting in poly (55.2 mol% 3HB-co-43.0 mol % D-phenyllactate-co-1.8 mol% D-mandelate) was prepared in an amount of 11.6% by weight of the dry cell weight (FIG. 8a, b), containing D-mandelate using D-mandelate as a substrate. Aromatic polymers have been successfully prepared.

Next, D-mandelate was produced in vivo by metabolic engineering. Amycolatopsis orientalis-derived hydroxymandelate synthetase (HmaS), S. coelicolor, hydroxymandelate oxidase in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA to produce D-mandelate from glucose (Hmo) and D-mandelate dehydrogenase (Dmd) from Rhodotorula graminis.

To construct pKM212-HmaS, the hydroxymandelic acid synthase gene (hmaS) from A. orientalis was used and the codon was cloned into a synthetic vector in Escherichia coli to construct plasmid pUC57-HmaSopt (GenScript, Piscataway, NJ, USA). ).

The pUC57-HmaSopt was linked to pKM212-MCS using restriction enzymes (EcoRI / KpnI). To construct pKM212-HmaSHmo, S. coelicolor's hydroxymandelate oxidase gene (hmo) was synthesized by codon-optimized hmo gene (GenScript, Piscataway, NJ, USA), and primers Hmo-F and Hmo-R were used. Amplified by PCR. The PCR product was linked to pKM212-HmaS using restriction enzymes (KpnI / BamHI) to prepare pKM212-HmaSHmo.

pUC57-Dmd containing the E. coli codon optimized dmd gene was synthesized to generate pKM212-HmaSHmoDmd (GenScript, Piscataway, NJ, USA) and E. coli codon optimized by PCR with primers Dmd-F and Dmd-R R. graminis D-mandelate dehydrogenase gene (dmd) was amplified. The PCR product was linked to pKM212-HmaSHmo using restriction enzymes (BamHI / SbfI) to prepare pKM212-HmaSHmoDmd.

PKM212-HmaSHmoDmd prepared above was introduced into E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA, to prepare a recombinant strain having mandelate production capacity.

As a result of culturing the recombinant strain having the above-described mandelyte production capacity in a medium containing 20 g / L glucose and 1 g / L sodium 3HB, 16.4 wt% poly (92.9 mol% 3HB-) of dry cell weight was obtained. co-6.3 mol% D-phenyllactate-co-0.8 mol% D-mandelate).

Aromatic polymer production was confirmed using 3-hydroxy-3-phenylpropionate (3HPh) as another possible aromatic monomer. Escherichia coli XB201TBAL strains were cultured in a medium containing 20 g / L glucose, 0.5 g / L 3-hydroxy-3-phenylpropionic acid and 1 g / L sodium 3HB to obtain a poly (33.3 mol% 3HB-co-18.0 mol% D-phenyl Lactate-co-48.7 mol% 3HPh) was confirmed to produce 14.7 wt% of the dry cell weight (Figure 8c, d). These results suggest that the system using the 2-hydroxyisocaproate-CoA transferase developed in the present invention can be widely used for the preparation of various aromatic polyesters.

Example 6: Preparation of Polyhydroxyalkanoates Containing Various Long Chain 2-HAs Using Recombinant Strains

In order to confirm that the system using the 2-hydroxyisocaproate-CoA transferase of the present invention can be used for the preparation of polyhydroxyalkanoate containing various long chain 2-HA, various long chain 2-HA Polymer production capability was confirmed using monomers [2-hydroxyisocaproate (2HIC), 2-hydroxyhexanoate (2HH) and 2-hydroxyoctanoate (2HO)] as monomers. Escherichia coli XL1-Blue expressing PhaC1437 and HadA was cultured in MR medium containing 1 g / L of 3HB, 20 g / L of glucose and concentration-specific (0.25, 0.5 and 1 g / L) long chain 2-HA. Copolymers containing hydroxyisocaproate, 2-hydroxyhexanoate or 2-hydroxyoctanoate were produced. In addition, it was confirmed that as the concentration of 2-HA contained in the medium increases, the mole fraction of the monomer contained in the copolymer increases (Table 4, Table 5 and Table 6).

Example 7 Preparation of Polyhydroxyalkanoates Containing Various Molar Fractions of Aromatic Monomers Through Synthetic Promoter-Based Flux Control

In order to prepare a strain producing aromatic PHA without external addition of 3HB, R. eutropha β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) were further expressed in XB201TBAL strain, and in glucose without 3HB supplementation. It was confirmed to produce aromatic PHA.

As a result, as expected, XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA resulted in poly (86.2 mol% 3HB-co-13.8 mol% D-phenyllactate) in MR medium at 18.0% by weight of dry cell weight. From 20 g / L glucose. In addition, the production of aromatic PHAs with various monomer mole fractions important for industrial applications was attempted by controlling the metabolic flux of PhaAB using a synthetic Anderson promoter (http://parts.igem.org/). Five different plasmids expressing PhaAB were constructed under five promoters of different intensities (SEQ ID NOs: 89-93) and introduced into XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA.

As PhaAB expression decreased, the mole fraction of D-phenyllactate monomer increased; Copolymers with D-phenyllactate of 11.0 mol%, 15.8 mol%, 20.0 mol%, 70.8 mol% and 84.5 mol%, respectively, could be prepared (FIGS. 9 a, b and table 7); By expressing PhaAB under the BBa_J23103 promoter, poly (15.5 mol% 3HB-co-84.5 mol% D-phenyllactate) was produced at 4.3% by weight of dry cell weight (FIG. 9B). These results suggest that by controlling the metabolic flux, aromatic polyesters with various aromatic monomer mole fractions can be produced.

Example 8 Preparation of Aromatic Polyhydroxyalkanoates Through Fed-Batch Fermentation

In this example, pH-stat culture of E. coli XB201TBAL strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters was performed without 3HB feeding. After 96 hours of incubation, poly (67.6 mol% 3HB-co-32.4 mol% D-phenyllactate) having a polymer content of 43.8 wt% of dry cell weight was produced at 2.5 g / L (FIG. 10C, d).

In addition, the ldhA gene of the E. coli XB201TBA chromosome was replaced with the fldH gene to further optimize the production of aromatic polyhydroxyalkanoate to optimize the gene expression system. In addition, the native promoter of the ldhA gene was replaced with a strong trc promoter to increase the expression of the fldH gene. A fed-batch fermentation was performed using a pulse feeding method to supply glucose. E. coli XB201TBAF strains expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437, and BBa_J23114 promoters are poly (69.1 mol% 3HB-co-38.1 mol% D-phenyllactate) having a polymer content of 55.0% by weight of dry cell weight. Was produced through fed-batch fermentation at 13.9 g / L (FIG. 10e, f), and the yield was 13.9 g / L in E. coli XB201TBAL strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters. It was 5.56 times higher than the yield of 2.5 g / L and much higher than that obtained by fed-batch culture of E. coli XB201TBAL strain expressing AroGfbr, PheAfbr, FldH, HadA, and PhaC1437 in medium supplemented with glucose and 3HB. These results can be successfully produced at high concentration through fed-batch culture of aromatic polyhydroxyalkanoate engineered strains (E. coli XB201TBAF strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters). Shows.

Example 9: Physical property analysis of polyhydroxyalkanoate containing aromatic monomer

Finally, the material properties of aromatic PHA produced by metabolically engineered Escherichia coli were investigated.

Polyhydroxyalkanoate (PHA) content and monomer composition were determined by GC or GC-MS. The collected cells were washed three times with distilled water, and then lyophilized for 24 hours, and the PHAs of the lyophilized cells were converted to the corresponding hydroxymethyl esters by acid catalyzed metanolysis. The resulting methyl ester was obtained using GC (Agilent 6890N, Agilent, USA) equipped with an Agilent 7683 autoinjector, frame ionization detector and fused silica capillary column (ATTM-Wax, 30 m, ID 0.53 mm, thickness 1.20 μm, Alltech, USA). Analyzed. The polymer was extracted by chloroform extraction and purified in cells using solvent extraction. The structure, molecular weight and thermal properties of the polymers were measured using nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and differential scanning calorimetry (DSC), respectively.

As a result, Figure 7 shows the results of the analysis of the poly (3HB-co-D-phenyl lactate) produced by E. coli XB201TBAL, Figure 8 shows the poly (3HB-co-D- produced by E. coli XB201TBAL Phenyllactate-co-D-mandelate) is shown.

Poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate) was amorphous, and the Tg increased significantly to 23.86 ° C. despite decreasing molecular weight as the mole fraction of D-phenyllactate in the copolymer increased It was. In addition, copolymers containing aromatics in the polymers reduced crystallinity. It is assumed that the aromatic ring of the polymer interferes with the crystallization of P (3HB) (derived by stereochemistry). In the case of P (3HB), it showed high brittleness due to the strong crystallinity, whereas in the resultant copolymer, the decrease of crystallinity and the improvement of Tg resulted in the improvement of mechanical toughness.

Although the present invention has been described in detail with reference to specific configurations, such description is directed to the preferred embodiments and will be apparent to those skilled in the art without limiting the scope of the invention. Thus, the substantial scope of the present invention will be defined by the claims and their equivalents.

According to the present invention, a biodegradable polymer containing an aromatic monomer or a long chain 2-HA as a monomer can be produced.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (32)

1. In microorganisms having the ability to produce acetyl-CoA from a carbon source, a gene encoding 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthetase are introduced, and an aromatic monomer or a long chain 2 A recombinant microorganism having a polyhydroxyalkanoate generating ability containing -HA as a monomer.
2. According to claim 1, wherein the polyhydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. Recombinant microorganisms characterized in that the PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from the following:

   An amino acid sequence comprising one or more variants 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: 2;

   Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

   Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

   The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).
3. The recombinant microorganism of claim 1, wherein the 2-hydroxyisocaproate-CoA transferase is hadA derived from Clostridium difficile 630.
4. The recombinant microorganism of claim 1 or 2, wherein the 2-hydroxyisocaproate-CoA transferase uses acetyl-CoA as a CoA donor.
5. The method of claim 1, wherein the aromatic monomer or long-chain 2-HA monomer is 2-hydroxyisocaproate, 2-hydroxyhexanoate, 2-hydroxyoctanoate, phenyllactate, 2-hydroxy- Recombinant microorganism, characterized in that selected from the group consisting of 4-phenylbutyrate, 3-hydroxy-3-phenylpropionate, 4-hydroxybenzoic acid and mandelate.
6. The recombinant microorganism according to claim 1, wherein a gene encoding β-ketothiolase and a gene encoding acetoacetyl-CoA reductase are further introduced.
7. A process for preparing polyhydroxyalkanoate containing as an monomer an aromatic monomer or a long chain 2-HA comprising the following steps:

   (a) culturing the recombinant microorganism of any one of claims 1 to 6 to produce a polyhydroxyalkanoate containing an aromatic monomer or a long chain 2-HA as a monomer; And

   (b) obtaining a polyhydroxyalkanoate containing the produced aromatic monomer or long chain 2-HA as a monomer.
8. The method for producing polyhydroxyalkanoate according to claim 7, wherein the recombinant microorganism is cultured in a medium containing an aromatic monomer or a long chain 2-HA as a monomer.
9. The method of claim 7, wherein the aromatic monomer or the long-chain 2-HA monomer is 2-hydroxyisocaproate, 2-hydroxyhexanoate, 2-hydroxyoctanoate, phenyl lactate, 2-hydroxy A process for producing polyhydroxyalkanoate, characterized in that it is selected from the group consisting of -4-phenylbutyrate, 3-hydroxy-3-phenylpropionate, 4-hydroxybenzoic acid and mandelate.
10. In microorganisms having the ability to produce acetyl-CoA from a carbon source, a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-deoxy-D-ara) The gene encoding the Vino-heptulsonate-7-phosphate) synthetase, the gene encoding the corismate mutase / prephenate dehydrogenase and the gene encoding the D-lactate dehydrogenase were introduced. And a recombinant microorganism having a polyhydroxyalkanoate-generating ability, containing phenyl lactate as a monomer.
11. The recombinant microorganism having polyhydroxyalkanoate-producing ability according to claim 10, wherein the 2-hydroxyisocaproate-CoA transferase is hadA derived from Clostridium difficile 630.
12. The method of claim 10, wherein the polyhydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. PHA synthase derived from a strain selected from the group consisting of 6-19 or a polyhydroxyalkanoate producing ability containing a phenyl lactate as a monomer, characterized in that the mutant enzyme of PHA synthase having an amino acid sequence selected below Recombinant Microorganisms:

    An amino acid sequence comprising one or more variants 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: 2;

    Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

    Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

    The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).
13. The gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding an amino acid sequence represented by SEQ ID NO: 11. A recombinant microorganism having a polyhydroxyalkanoate-generating ability, containing a phenyl lactate as a monomer.
14. The poly-containing monomer of phenyl lactate according to claim 10, wherein the gene encoding the corismate mutase / prephenate dehydrogenase is a gene encoding an amino acid sequence represented by SEQ ID NO: 9. Recombinant microorganism having hydroxyalkanoate producing ability.
15. The polyhydroxyalkano containing a phenyl lactate as a monomer according to claim 10, wherein the gene encoding the D-lactate dehydrogenase is a gene encoding an amino acid sequence represented by SEQ ID NO: 10. Recombinant microorganisms having the ability to produce.
16. The recombinant microorganism having a polyhydroxyalkanoate-producing ability with phenyllactate as a monomer according to claim 10, wherein the gene encoding the D-lactate dehydrogenase is a fldH gene.
17. The recombinant microorganism having a polyhydroxyalkanoate-producing ability according to claim 10, wherein a gene encoding β-ketothiolase and a gene encoding acetoacetyl-CoA reductase are further introduced.
18. The tyrR gene, a gene encoding pyruvate oxidase, a gene encoding pyruvate formate lyase, a gene encoding acetaldehyde dehydrogenase, a gene encoding fumarate reductase, a tyrosine aminotransferase A recombinant microorganism having a polyhydroxyalkanoate-producing ability containing a phenyl lactate as a monomer, wherein at least one gene selected from the group consisting of a gene encoding the gene and a gene encoding aspartic acid aminotransferase is deleted.
19. A method for preparing polyhydroxyalkanoate containing phenyl lactate as a monomer, comprising the following steps;

    (a) culturing the recombinant microorganism of any one of claims 10 to 18 to produce a polyhydroxyalkanoate containing phenyllactate as a monomer; And

    (b) obtaining a polyhydroxyalkanoate containing the produced phenyl lactate as a monomer.
20. In microorganisms having the ability to produce acetyl-CoA from a carbon source, a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-deoxy-D-ara) Gene encoding the Vino-heptulsonate-7-phosphate) synthetase, gene encoding corismate mutase / prephenate dehydrogenase and gene encoding D-lactate dehydrogenase, hydroxy A gene encoding mandelate synthase, a gene encoding hydroxymandelate oxidase, and a gene encoding D-mandelate dehydrogenase are introduced, and polyhydroxyalkanoate containing mandelate as a monomer Recombinant microorganisms having the ability to produce.
21. 21. The recombinant microorganism having polyhydroxyalkanoate producing ability according to claim 20, wherein the 2-hydroxyisocaproate-CoA transferase is hadA derived from Clostridium difficile 630.
22. The method of claim 20, wherein the polyhydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. PHA synthase derived from a strain selected from the group consisting of 6-19 or a mutant enzyme of PHA synthase having an amino acid sequence selected from the following. Recombinant having polyhydroxyalkanoate-producing ability containing a mandelate as a monomer microbe:

    An amino acid sequence comprising one or more variants 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: 2;

    Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;

    Amino acid sequence (C1310) wherein E130D, S477F and Q481K are mutated in the amino acid sequence of SEQ ID NO: 2; And

    The amino acid sequence of E130D, S477F and Q481R having a mutated amino acid sequence of SEQ ID NO: 2 (C1312).
23. The gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding an amino acid sequence represented by SEQ ID NO: 22. A recombinant microorganism having a polyhydroxyalkanoate generating ability containing a mandelate as a monomer.
24. The polyhydrate containing mandelate as a monomer according to claim 20, wherein the gene encoding the corismate mutase / prephenate dehydrogenase is a gene encoding an amino acid sequence represented by SEQ ID NO: 9. Recombinant microorganism having the ability to produce oxyalkanoate.
25. The polyhydroxyalkanoate containing mandelate as a monomer according to claim 20, wherein the gene encoding the D-lactate dehydrogenase is a gene encoding the amino acid sequence represented by SEQ ID NO: 10. Recombinant microorganisms having the ability to produce.
26. 21. The polyhydroxyalkanoate-producing ability according to claim 20, wherein the gene encoding the hydroxymandelate synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 11. Recombinant microorganisms.
27. 21. The polyhydroxyalkanoate-producing ability of claim 20, wherein the gene encoding the hydroxymandelate oxidase is a gene encoding the amino acid sequence represented by SEQ ID NO: 12. Recombinant microorganisms.
28. 21. The polyhydroxyalkanoate-producing ability of claim 20, wherein the gene encoding the D-mandelate dehydrogenase is a gene encoding the amino acid sequence represented by SEQ ID NO: 13. Branched recombinant microorganism.
29. The recombinant microorganism having polyhydroxyalkanoate-producing ability according to claim 20, wherein the gene encoding the D-lactate dehydrogenase is a fldH gene.
30. 21. The polyhydroxyalkano containing monomer as a monomer according to claim 20, wherein a gene encoding β-ketothiolase and a gene encoding acetoacetyl-CoA reductase are further introduced. Recombinant microorganisms having the ability to produce.
31. A method for preparing polyhydroxyalkanoate containing mandelate as a monomer comprising the following steps;

    (a) culturing the recombinant microorganism of any one of claims 20 to 30 to produce a polyhydroxyalkanoate containing mandelate as a monomer; And

    (b) obtaining a polyhydroxyalkanoate containing the produced mandelate as a monomer.
32. Method for producing polyhydroxyalkanoate containing as monomer a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate ;

    (a) The recombinant microorganism of any one of claims 1 to 6 contains a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate. Culturing in a medium; And

    (b) obtaining a polyhydroxyalkanoate containing as a monomer a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate.

Images