Mutants of pha synthase derived from pseudomonas 6-19 and methods of making lactic acid ester homopolymers or copolymers using the mutants

Abstract

The present invention relates to a polyhydroxyalkanoate synthase (PHA synthase) mutant derived from Pseudomonas 6-19 (KCTC 11027BP) which can produce a lactate ester polymer and / or copolymer by using lactyl-CoA as a substrate. The present invention relates to a method for producing a lactate ester polymer and / or copolymer using the said synthase mutant. The polyhydroxyalkanoate synthase mutant of the present invention derived from Pseudomonas 6-19 can effectively produce a lactate ester polymer and / or copolymer by using lactyl-CoA as a substrate which is difficult to use as a substrate with a conventional polyhydroxyalkanoate synthase.

Description

[0001] This application is a divisional application of the invention patent application entitled "A mutant of PHA synthase derived from Pseudomonas 6-19 and a method for preparing lactate homopolymers or copolymers using the mutant". The national application number of the parent application is "200780043377.3", the PCT international application date is November 21, 2007, and the PCT international application number is PCT / KR2007 / 005858. Technical Field

[0002] This invention relates to a polyhydroxyalkanoate synthase mutant derived from Pseudomonas sp. 6-19, wherein the mutant can be used as a substrate to produce lactate polymers and / or lactate copolymers. The invention also relates to a method for preparing lactate polymers and / or lactate copolymers, wherein the method uses the aforementioned polyhydroxyalkanoate synthase mutant. Background Technology

[0003] Polylactic acid ester (PLA) is a common biodegradable polymer derived from lactate esters, with various applications as a general or medical polymer. Currently, PLA is prepared by polymerizing lactate esters produced by microbial fermentation; however, direct polymerization of lactate esters only yields low molecular weight PLA (1000-5000 Daltons). To synthesize high molecular weight (>100,000 Daltons) PLA, a method can be used to polymerize low molecular weight PLA obtained from the direct polymerization of lactate esters via a chain coupling agent. However, this method has the following drawbacks: the preparation of high molecular weight PLA becomes complex due to the addition of solvents or chain coupling agents, and it is not easy to remove these solvents or chain coupling agents. Currently, among the methods for preparing commercially available high molecular weight PLA, a method is being used in which lactate esters are converted into lactate esters to synthesize PLA through cyclization and dehydration of the lactate ring.

[0004] PLA homopolymers can be readily obtained by chemical synthesis using lactate esters, but it is difficult to prepare lactate ester copolymers with multiple monomer units, and their commercial availability is very low.

[0005] Meanwhile, polyhydroxyalkanoates (PHAs) are polyesters that accumulate in microorganisms as carbon and energy storage compounds when other nutrients such as phosphorus, nitrogen, magnesium, and oxygen are lacking but carbon sources are abundant. Because PHAs share similar properties with petroleum-derived synthetic polymers and also exhibit excellent biodegradability, they are considered an alternative to synthetic plastics.

[0006] To produce PHA in microorganisms, an enzyme that converts microbial metabolites into PHA monomers and a PHA synthase that synthesizes PHA polymers from the PHA monomers are required. The same system is needed when using microorganisms to produce PLA and lactate copolymers, and in addition to providing hydroxyl-CoA (the original substrate for PHA synthase), an enzyme capable of providing lactyl-CoA is also required.

[0007] Therefore, the inventors of this invention have developed a system for providing lactyl-CoA using a propionyl-CoA transferase derived from *Clostridium propionicum*, and have successfully prepared PLA and lactate copolymers (Korean Patent Application Publication No. 10-2006-0121555). However, it exhibits low PHA synthase activity for hydroxyalkyl esters hydroxylated at the 2-position. PHA synthase activity against lactyl-CoA has been reported in vitro, but such activity has been reported to be very low, as described above (Zhang et al., *Appl. Microbiol. Biotechnol.*, 56:131, 2001; Valentin and Steinbuchel, *Appl. Microbiol. Biotechnol.*, 40:699, 1994). Yuan et al., Arch Biochem Biophys., 394:87, 2001. Therefore, if PHA synthase cannot efficiently utilize lactyl-CoA and is used to produce PLA and lactate copolymers, the synthesis efficiency will inevitably be low. That is to say, because lactate (hydroxyalkyl esters hydroxylated at the 2-carbon position) is not a suitable substrate for PHA synthase, a PHA synthase that can efficiently utilize lactyl-CoA is crucial for the efficient synthesis of PLA and lactate copolymers. Summary of the Invention

[0008] Technical issues

[0009] Therefore, one object of the present invention is to provide a PHA synthase that can effectively use lactoyl-CoA as a substrate.

[0010] Another object of the present invention is to provide a method for preparing PLA and lactate copolymers, wherein the method uses cells or plants containing PHA synthase genes and propionyl-CoA transferase genes capable of using lactyl-CoA as a substrate.

[0011] Technical solution

[0012] To achieve the aforementioned objective, the present invention provides a polyhydroxyalkanoate synthase mutant that uses lactyl-CoA as a substrate to produce lactate polymers or lactate copolymers and has the amino acid sequence of SEQ.ID No:10, wherein the glutamine at position 481 of the amino acid sequence of SEQ.ID No:10 is mutated.

[0013] Preferably, the present invention provides a polyhydroxyalkanoate synthase mutant, wherein at least one amino acid selected from glutamic acid at position 130, serine at position 325, and serine at position 477 is further mutated.

[0014] More preferably, the present invention provides a polyhydroxyalkanoate synthase mutant that uses lactyl-CoA as a substrate to produce lactate polymers or lactate copolymers and has the amino acid sequence of SEQ.ID No:10, wherein the amino acid sequence has any of the following mutations:

[0015] a) S325T and Q481M;

[0016] b) E130D and Q481K;

[0017] c) S325T and Q481K;

[0018] d) E130D and Q481M;

[0019] e) E130D and Q481R;

[0020] f) E130D, S325T and Q481M;

[0021] g)E130D, S325T and Q481K;

[0022] h)E130D, S477R and Q481K;

[0023] i) E130D, S477R and Q481M;

[0024] j)E130D, S477R and Q481R;

[0025] k)E130D, S477H and Q481K;

[0026] l)E130D, S477H and Q481M;

[0027] m)E130D, S477H and Q481R;

[0028] n)E130D, S477F and Q481K;

[0029] o)E130D, S477F and Q481M;

[0030] p)E130D, S477F and Q481R;

[0031] q)E130D, S477Y and Q481K;

[0032] r)E130D, S477Y and Q481M;

[0033] s)E130D, S477Y and Q481R;

[0034] t)E130D, S325T, S477R and Q481M;

[0035] u)E130D, S325T, S477R and Q481K;

[0036] v)E130D, S325T, S477F and Q481M;

[0037] w)E130D, S325T, S477G and Q481M; or

[0038] x)E130D, S325T, S477F and Q481K.

[0039] The inventors of this invention have demonstrated that some mutants of the polyhydroxyalkanoate synthase of Pseudomonas 6-19 can use lactyl-CoA as a substrate and produce lactate polymers and / or copolymers very efficiently, thereby completing this invention.

[0040] The present invention also provides a gene encoding the polyhydroxyalkanoate synthase mutant.

[0041] The present invention further provides a recombinant vector containing genes for synthesizing lactate polymers or copolymers.

[0042] More preferably, the present invention provides a recombinant vector that further comprises a gene encoding propionyl-CoA transferase (pct).

[0043] The present invention also provides cells or plants transformed using the above-described recombinant vector.

[0044] The present invention also provides cells or plants obtained by transformation using the above-described recombinant vector, wherein the original cells or plants do not have a gene encoding propionyl-CoA transferase.

[0045] The present invention also provides a method for preparing lactate polymers or copolymers, wherein the method includes culturing the cells or plants.

[0046] More preferably, the present invention provides the method wherein the culture is carried out in a culture medium containing 3-hydroxybutyrate (3-HB) and the copolymer prepared is a copolymer containing 3-hydroxybutyrate monomer units and lactate monomer units.

[0047] The term “copolymer” as used herein is intended to include binary copolymers consisting of two different monomers, ternary copolymers consisting of three different monomers, or quaternary copolymers consisting of four different monomers.

[0048] In this invention, the hydroxyalkanoate is at least one selected from the following compounds: 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, medium-chain (C) ester, and hydroxyalkanoates. 6～14(D)-3-hydroxycarboxylic acid, 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4-hydroxyvalerate, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5-hydroxyvalerate, 5-hydroxyhexanoic acid, 6-hydroxydodecanoic acid, 3-hydroxy-4-pentenoic acid, 3-hydroxy-4-trans-hexenoic acid, 3-hydroxy-4-cis-hexenoic acid, 3-hydroxy-5-hexenoic acid, 3-hydroxy-6-trans-octenanoic acid, 3-hydroxy-6-cis-octenanoic acid, 3-hydroxy-7-octenanoic acid, 3-hydroxy-8-nonenoic acid, 3-hydroxy -9-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-hydroxy-6-cis-dodecenoic acid, 3-hydroxy-5-cis-tetradecenoic acid, 3-hydroxy-7-cis-tetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylpentanoic acid, 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methylhexanoic acid, 3-hydroxy-6-methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-7- Methyl-6-octenic acid, malic acid, methyl 3-hydroxysuccinate, methyl 3-hydroxyhexanoate, methyl 3-hydroxyoctanoate, methyl 3-hydroxyazelate, methyl 3-hydroxysepiacetate, ethyl 3-hydroxyoctanoate, ethyl 3-hydroxysepiacetate, propyl 3-hydroxyheptanate, benzyl 3-hydroxysepiacetate, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvalerate, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, p-cyanophenoxy-3-hydroxybutyric acid, p-cyanophenoxy-3-hydroxyvalerate, p-cyanophenoxy-3-hydroxyhexanoic acid, p-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvalerate, 3-hydroxy-5 ... Cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-6,7-epoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8-chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid, 3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid, and 3-hydroxy-2,6-Dimethyl-5-heptenic acid.

[0049] The present invention also provides a method for preparing a lactate polymer or a lactate copolymer, wherein the method includes culturing the cells or plants.

[0050] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence capable of expressing that DNA within a suitable host. In this invention, the vector can be a plasmid, a bacteriophage, or a simple genomic insert. When used to transform a suitable host, the vector can self-replicate and operate regardless of the host genome, or in some cases, the vector may be incorporated into the host genome. Because plasmids are currently the most commonly used vector, plasmids may be used alternatively to vectors herein. However, vectors according to the invention include different types of vectors having the same function, which are known or will be known to those skilled in the art.

[0051] The term "expression control sequence" refers to the DNA sequence necessary for expressing an operable linked coding sequence in a specific host. This control sequence includes: a promoter for transcription; an arbitrary operator gene sequence for controlling transcription; a sequence encoding a ribosome-binding site suitable for mRNA; and a sequence for controlling the termination of transcription and translation. For example, control sequences suitable for prokaryotes include a promoter, an arbitrary operator gene sequence, and a ribosome-binding site. For eukaryotes, they include a promoter, a polyadenylated nucleotide signal, and an enhancer. In plasmids, the promoter is one of the most significant factors affecting gene expression levels. Preferably, the SRα promoter, promoters derived from cytomegalovirus, etc., are used as promoters for high expression.

[0052] Any of the various expression control sequences can be used to express the DNA sequence of the present invention in a vector. Examples of useful expression control sequences include early or late promoters of SV40 or adenovirus, lac systems, trp systems, TAC or TRC systems, T3 and T7 promoters, major operator genes and promoter regions of bacteriophage λ, control regions of fd-encoded proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for phosphatases such as Pho5, promoters of yeast α-mating systems, and known structural sequences controlling gene expression in eukaryotes, prokaryotes, or viruses thereof, and various combinations thereof.

[0053] When a nucleic acid is placed into a functional relationship with other nucleic acid sequences, it is "operably linked." This can mean the way a gene and a control sequence are linked, because gene expression is possible when a suitable molecule (e.g., a transcriptional activator protein) binds to the control sequence. For example, when the DNA of a current peptide sequence or secretory leader is expressed as a preprotein involved in the secretion of the polypeptide, it is operably linked to the DNA of that polypeptide; when a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to that coding sequence; or when a ribosome binding site is set to facilitate translation, it is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are adjacent, and in the case of a secretory leader, adjacent and within the reading phase. However, enhancers do not need to be adjacent. Linkage is achieved by joining at convenient restriction sites. If these sites are not available, synthetic oligonucleotide adapters or linkers are used according to standard practice.

[0054] The term "expression vector" as used here generally refers to a double-stranded DNA fragment that serves as a recombinant vector, into which a typical foreign DNA fragment is inserted. The foreign DNA refers to heterologous DNA that is not naturally occurring in the host cell. Once the expression vector is introduced into the host cell, it can replicate regardless of the host chromosomal DNA, producing several copies and inserting the foreign DNA into them.

[0055] As is known to those skilled in the art, in order to increase the expression level of a gene transfected into a host cell, the corresponding gene should be operatively linked to a sequence for controlling transcription and decoding expression, which functions in the selected expression host. Preferably, the expression control sequence and the corresponding gene are contained in an expression vector that also contains a viral selection marker and an origin of replication. If the expression host is a eukaryote, the expression vector should further contain expression markers useful in eukaryotic expression hosts.

[0056] In this invention, various vectors, such as plasmid vectors, phage vectors, granular vectors, or YAC (yeast artificial chromosome) vectors, can be used as the aforementioned vectors. For the purposes of this invention, plasmid vectors are preferred. Typical plasmid vectors that can be used for these purposes have: (a) an origin of replication so that it leads to efficient replication such that each host cell contains several hundred copies of the plasmid vector; (b) an antibiotic resistance gene so that host cells transformed with the plasmid vector can be selected; and (c) a sequence containing a restriction enzyme site into which the foreign DNA fragment will be inserted. Even in the absence of a suitable restriction enzyme site, the vector or foreign DNA can be readily ligated using synthetic oligonucleotide adapters or linkers according to conventional methods.

[0057] The recombinant vector of the present invention can be transformed into suitable host cells according to methods known in the art. Preferred host cells in the present invention are prokaryotic cells, more preferably *Escherichia coli*. Preferred *E. coli* strains include: *E. coli* DH5a, *E. coli* JM101, *E. coli* K12, *E. coli* W3110, *E. coli* X1776, *E. coli* XL1-Blue (Stratagene), and *E. coli* B. However, other *E. coli* strains and other prokaryotic cell species, such as FMB101, NM522, NM538, and NM539, can also be used. In addition to the aforementioned *E. coli*, various *Agrobacterium* species, such as *Agrobacterium* A4, various *Bacillus* species, such as *Bacillus subtilis*, various enterobacteria, such as *Salmonella typhimurium* or *Serratia marcescens*, and various *Pseudomonas* species can also be used as host cells, but the scope of the present invention is not limited to the above examples.

[0058] Alternatively, prokaryotic cell transformation can be readily performed using the calcium chloride method described in section 1.82 of Sambrook et al. (supra). Alternatively, these cell types can be transformed using electroporation (Neumann et al., EMBO J., 1:841 (1982)).

[0059] Transformation of plants containing transferase and synthase genes can be achieved using conventional methods employing Agrobacterium or viral vectors. For example, transformed plants can be obtained by transforming Agrobacterium with a recombinant vector containing the genes of this invention and then infecting tissues of the target plant with the transformed Agrobacterium. More specifically, transformed plants can be prepared by the following steps: (a) pre-culturing explants of the target plant and then transforming the explants by co-culturing the explants with transformed Agrobacterium; (b) culturing the infected explants to induce callus; and (c) cutting off the obtained callus and culturing it in a shoot-inducing medium.

[0060] As used herein, the term "explant" refers to a tissue fragment cut from a plant and includes cotyledons or hypocotyls. Cotyledons or hypocotyls can be used as explants in this invention. More preferably, cotyledons obtained by germinating plant seeds in MS medium after sterilization and washing are used.

[0061] Plants that can be used for transformation in this invention include, but are not limited to, tobacco, tomato, pepper, beans, rice, and corn. Furthermore, even if the transformed plant is sexually reproduced, it will be apparent to those skilled in the art that such a plant can also be propagated asexually using methods such as plant tissue culture. Attached Figure Description

[0062] Figure 1This is a diagram showing the preparation method of a recombinant expression vector containing the polyhydroxyalkanoate synthase gene derived from Pseudomonas 6-19.

[0063] Figure 2 This is under conditions where PHB can be synthesized, and in a culture medium containing phaCl. Ps6-19 synthase and SCL mutant (phaC1) Ps6-19 200 and phaC1 Ps6-19 FACS (Fluorescence-activated cell sorting) results of E. coli transformed with recombinant vectors (pPs619C1-ReAB, pPs619C1200-ReAB and pPs619C1300-ReAB).

[0064] Figure 3 This is a simplified diagram of a constitutive expression vector that simultaneously expresses PHA synthase and CP-PCT.

[0065] Figure 4 This is a diagram showing the preparation method of a recombinant expression vector containing the PHA synthase gene, SCL mutant gene, and CP-PCT gene derived from Pseudomonas 6-19. Detailed Implementation

[0066] The present invention will now be described in more detail. The embodiments provided below are illustrative to help those skilled in the art understand the invention, but are not intended to limit the scope of the invention.

[0067] In particular, although the following examples disclose the synthesis of poly(3-hydroxybutyrate-co-lactic acid ester) (P(3HB-co-LA)) obtained by adding 3-hydroxybutyrate (3-HB) when preparing lactate copolymers with PHA synthase mutants, it will be apparent to those skilled in the art that various copolymers containing different hydroxyalkanoates and lactates can be prepared by adding other hydroxyalkanoates other than 3-HB.

[0068] <Example 1> Cloning and construction of expression vector of PHA synthase gene derived from Pseudomonas 6-19

[0069] To isolate PHA synthase (phaC1) derived from Pseudomonas 6-19 (KCTC 11027BP) Ps6-19 ) gene, extract all DNA from Pseudomonas 6-19, and according to phaC1 Ps6-19The gene sequence was prepared using primers for SEQ ID NO:1 and 2 (Ae-jin Song, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004) and PCR was performed to obtain phaC1. Ps6-19 The genes.

[0070] SEQ ID NO:1:5-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3

[0071] SEQ ID NO:2:5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3

[0072] As a result of detecting PCR reaction products by agarose gel electrophoresis, corresponding to phaC1 was observed. Ps6-19 A 1.7Kbp gene fragment. For the expression of phaC1. Ps6-19 Synthase, constructing the operon of the constitutive expression system of the supplying monomer enzyme and the synthase. Figure 1 ).

[0073] A DNA fragment containing the PHB-synthesis operon derived from Ralstonia eutropha H16 was excised from the pSYL105 vector using BamHI / EcoRI (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347) and inserted into the BamHI / EcoRI recognition site of pBluescript II (Stratagene) to construct the pReCAB recombinant vector.

[0074] It is known that the pReCAB vector constitutively expresses PHA synthase (phaC) via the PHB operon promoter. RE ) and the enzyme that supplies monomers (phaA) RE and phaB RE It is also known to work well in *E. coli* (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347). The pReCAB vector was cleaved with BstBI / SbfI to delete *R. eutrophus* H16PHA synthase (phaC). RE Then, the above phaC1 Ps6-19 Gene insertion into the BstBI / SbfI site to prepare the pPs619C1-ReAB recombinant vector ( Figure 1 ).

[0075] phaC1 with two BstBI / SbfI sites at both ends was prepared by removing the internal BstBI site using the SDM (site-directed mutagenesis) method without amino acid mutations. Ps6-19 The synthase gene fragment was extracted, and overlap PCR was performed using primers SEQ ID NO:3 and 4, SEQ ID NO:5 and 6, and SEQ ID NO:7 and 8 to add the BstBI / SbfI-recognition site.

[0076] SEQ ID NO:3:5-atg ccc gga gcc ggt tcg aa-3

[0077] SEQ ID NO:4:5-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC-3

[0078] SEQ ID NO:5:5-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3

[0079] SEQ ID NO:6:5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3

[0080] SEQ ID NO:7:5-GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3

[0081] SEQ ID NO:8:5-aac ggg agg gaa cct gca gg-3

[0082] The phaC1 of the pPs619C1-ReAB recombinant vector was verified by sequencing. Ps6-19 The gene sequence is shown in SEQ ID NO:9, and the amino acid sequence encoded by it is shown in SEQ ID NO:10.

[0083] Similarity tests of these sequences showed that this gene shares 84.3% nucleotide sequence identity and 88.9% amino acid sequence identity with phaC1 derived from Pseudomonas strain 61-3 (Matsusaki et al., J. Bacteriol., 180:6459, 1998), thus confirming that the two synthases are highly similar. These results confirm that the phaC1 obtained in this invention... Ps6-19 The synthase is type II PHA synthase.

[0084] To confirm phaC1 Ps6-19 To determine whether PHB synthesis was achieved, *E. coli* XL-1Blue (Stratagene) was transformed with the pPs619C1-ReAB recombinant vector and cultured in PHB detection medium (LB agar, 20 g / L glucose, 0.5 μg / ml Nile Red). The result was that no PHB synthesis was observed.

[0085] <Example 2> Preparation of substrate-specific mutants of PHA synthase derived from Pseudomonas 6-19

[0086] Among various types of PHA synthases, type II PHA synthases are known to be MCL-PHA (medium-chain long PHA) synthases, capable of polymerizing substrates with relatively long carbon chains. This MCL synthase is expected to be used in the preparation of lactate polymers. Even the phaC1 synthase derived from Pseudomonas 61-3 (which is similar to the phaC1 of this invention)... Ps6-19 While phaC1 synthases exhibit high homology, they are type II synthases. However, phaC1 synthases have been reported to possess a relatively broad range of substrate specificity (Matsusaki et al., J. Bacteriol., 180:6459, 1998), and studies have been reported on mutants suitable for preparing SCL-PHA (short-chain long PHA) (Takase et al., Biomacromolecules, 5:480, 2004). Based on these results, sequence comparison analysis identified three amino acid positions affecting SCL activity, and phaC1 synthases (shown in Table 1 below) were prepared using primers with SEQ ID NO:11–14 via the SDM method. Ps6-19 Synthase mutant.

[0087] Table 1

[0088]

[0089]

[0090] SEQ ID NO:11:5-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC-3

[0091] SEQ ID NO:12:5-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG-3

[0092] SEQ ID NO:13:5-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC-3

[0093] SEQ ID NO:14:5-GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG-3

[0094] SEQ ID NO:15:5-atc aac ctc atg acc gat gcg atg gcg ccg acc-3

[0095] SEQ ID NO:16:5-ggt cgg cgc cat cgc atc ggt cat gag gtt gat-3

[0096] These recombinant vectors were used to transform *E. coli* XL-1Blue, which was then cultured in PHB detection medium (LB agar, glucose 20 g / L, Nile red 0.5 ug / ml). PHB synthesis was observed in both *E. coli* XL-1Blue transformed with pPs619C1200-ReAB and pPs619C1300-ReAB. That is, using phaA... RE and phaB RE The enzyme supplied the monomer prepared 3HB-coenzyme A from glucose and then processed it via phaC1. Ps6-19 Synthase SCL mutant (phaC1) Ps6-19 200 and phaC1 Ps6-19 300) PHB was prepared using 3HB-CoA as a substrate. For quantitative analysis, recombinant *E. coli* XL1-Blue transformed bacteria were cultured for 4 days at 37°C in LB medium containing glucose (20 g / L). The cultured recombinant *E. coli* were subjected to sucrose shock and stained with Nile Red, and analyzed using FACS (Fluorescence Activated Cell Sorting). Figure 2 ).

[0097] E. coli XL1-Blue transformed with the pPs619C1-ReAB vector containing wild-type synthase was not stained with Nile Red, while E. coli XL1-Blue transformed with pPs619C1200-ReAB or pPs619C1300-ReAB showed high fluorescence due to the presence of PHB stained with Nile Red in the cells. Furthermore, the content of PHB prepared in the cells was evaluated by collecting cultured microorganisms, centrifuging them, drying them in an 80°C desiccator for 48 hours, and then performing gas chromatography analysis. The PHB content in E. coli XL1-Blue transformed with pPs619C1200-ReAB and pPs619C1300-ReAB was 29.7% (w / w) and 43.1% (w / w), respectively, while no PHB was detected in the case of pPs619C1-ReAB.

[0098] <Example 3> Construction of recombinant Escherichia coli capable of expressing PHA synthase and propionyl-CoA transferase derived from Pseudomonas 6-19, and preparation of PLA or lactate copolymers using it.

[0099] In this embodiment, propionyl-CoA transferase derived from Clostridium propionitum (CP-PCT) is used to supply lactyl-CoA, a monomer required for the synthesis of PLA and lactate copolymers. Figure 3 The operon for constructing a constitutive expression system simultaneously expressing PHA synthase and CP-PCT is shown. The toxicity of CP-PCT to microorganisms is well-known. That is, in IPTG-induced tac promoter or T7 promoter expression systems (widely used for recombinant protein expression), all microorganisms die rapidly after the addition of the inducer. Therefore, it is considered appropriate to use an expression system in which the expression is weaker but continuous according to microbial growth. The fragment obtained by PCR using Clostridium propionitum chromosomal DNA and primers of SEQ ID NO:17 and 18 was used as cp-pct, and the NdeI site of wild-type CP-CPT was removed by SDM for easy cloning. Figure 4 ).

[0100] SEQ ID NO:17:5-ggaattcATGAGAAAGGTTCCCATTATTACCGCAGATGA

[0101] SEQ ID NO:18:5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gccttc tg

[0102] In addition, overlapping PCR was performed using primers from SEQ ID NO:19 and 20 to add the SbfI / NdeI recognition site.

[0103] SEQ ID NO:19:5-agg cct gca ggc gga taa caa ttt cac aca gg-3

[0104] SEQ ID NO:20:5-gcc cat atg tct aga tta gga ctt cat ttc c-3

[0105] Cut the phaC1-containing section with SbfI / NdeI. Ps6-19 Synthase SCL mutant (phaC1) Ps6-19 The pPs619C1300-ReAB vector (300) was used to remove the enzyme (phaA) from the donor monomer derived from *Ralstonia eutropha* H16. RE and phaB RE Then, the cp-pct gene of the PCT clone was inserted into the SbfI / NdeI site to prepare the pPs619C1300-CPPCT recombinant vector. Figure 4 ).

[0106] In addition, other phaC1-containing preparations were prepared using the same method described above. Ps6-19 Synthase SCL mutant (phaC1) Ps6-19 200) pPs619C1200-CPPCT recombinant vector and containing wild-type phaC1 Ps6-19 The pPs619C1-CPPCT recombinant vector for synthase. To confirm whether cp-pct prepared the polymer via supplied monomers, *E. coli* XL-1Blue was transformed with the pPs619C1200-CPPCT and pPs619C1300-CPPCT recombinant vectors and cultured in PHB assay medium (LB agar, glucose 20 g / L, 3HB 2 g / L, Nile red 0.5 μg / ml). The results of this assay showed that no PHB synthesis was observed in either vector.

[0107] PLA and lactate copolymers were prepared by glass bottle culture under various conditions using the pPs619C1300-CPPCT recombinant vector. The results are shown in Table 2 below.

[0108] Table 2

[0109]

[0110] Lactate is produced intracellularly by performing the two-step culture process by replacing the culture medium with MR medium followed by anaerobic culture. As a result of culturing recombinant Escherichia coli under various conditions, the prepared PLA homopolymer was approximately 1% based on the weight of dried cells, and the lactate copolymer prepared by adding 3HB as a substrate was approximately 6%. The composition of the MR medium used in fed-batch culture according to the invention is shown in Table 3 below.

[0111] Table 3

[0112]

[0113]

[0114] * Trace components ( / L): FeSO4·H2O, 10g; ZnSO4·H2O, 2.25g; CuSO4·H2O, 1g; MnSO4·H2O, 0.5g; CaCl2·H2O, 2g; Na2B4O7·H2O, 0.23g; (NH4)6Mo7O 24 0.1g; 35% HCl, 10mL.

[0115] <Example 4> Preparation of P(3HB-co-LA) copolymer by fed-batch culture of recombinant Escherichia coli

[0116] Recombinant *E. coli* (transformed with the pPs619C1300-CPPCT vector) was cultured in 3 mL of LB medium containing 20 g / L glucose, 100 mg / L ampicillin, etc., at 37°C for 12 hours with stirring at 200 rpm. This culture was then inoculated into 100 mL of the same medium and cultured under the same conditions for 6 hours. The final medium was used as the seed culture for fed-batch culture. MR medium was used as the starting medium for fed-batch culture. Fed-batch culture was initiated by inoculating 100 mL of the seed culture into 2.4 L of MR medium. The medium temperature was 37°C, the pH was adjusted to 6.8–6.9 with a 14% ammonia solution, and dissolved oxygen was maintained at more than 20% above saturation by controlling the air supply and stirring speed. The air supply rate was 1 vvm. When the glucose in the initial culture medium was depleted, 20 g of glucose and 5 g of 3-hydroxybutyrate (3-HB) were added, and the stirring speed was adjusted to 200 rpm and the air supply rate reduced to 0.1 vvm to change the aerobic conditions to anaerobic conditions. Glucose was supplied five times throughout the culture. During these five supply cycles, 5 g of 3-HB was supplied simultaneously on the first and third cycles. After terminating the culture, the cells were collected by centrifugation and freeze-dried.

[0117] To purify the prepared polymer, it was extracted from the freeze-dried cells using chloroform via a Soxhlet extractor. Most of the chloroform was then removed from the chloroform solution containing the polymer using a rotary evaporator, and methanol was added to the remaining solution to precipitate the polymer. The precipitated polymer was filtered off and dried in a vacuum desiccator for 12 hours.

[0118] In addition, a portion of the cells obtained by centrifugation were dried in a desiccator at 80°C for 48 hours, and the content of P(3HB-co-LA) copolymer in the cells was analyzed by gas chromatography. PLA homopolymer and P(3HB-co-3HV) copolymer containing 12 wt% 3HV were used as standards.

[0119] As a result, based on the weight of the dried cells, the content of P(3HB-co-LA) synthesized in E. coli was approximately 10%, while the content of PLA in the copolymer was 88 mol%.

[0120] This example, involving gas chromatography analysis of the final polymer, demonstrates that the obtained polymer is a P(3HB-co-LA) copolymer with a PLA content of 88 mol%.

[0121] <Example 5> Preparation of PLA homopolymer by fed-batch culture of recombinant Escherichia coli

[0122] Following the method of Example 4, seed culture of recombinant *E. coli* transformed with the pPs619C1300-CPPCT vector was performed, and fed-batch culture was initiated by inoculating 100 mL of seed culture into 2.4 L of MR medium. The medium temperature was 37°C, and the pH was adjusted to 6.8-6.9 with a 14% ammonia solution. Dissolved oxygen was maintained at more than 20% above saturation air by controlling the air supply and stirring speed. At this time, the air supply rate was 1 vvm. When the glucose contained in the initial medium was depleted, 20 g of glucose was added, and the stirring speed was adjusted to 200 rpm while the air supply rate was reduced to 0.1 vvm to change the aerobic conditions to anaerobic conditions. Glucose was supplied 5 times for the entire culture. After the culture was terminated, the cells were collected by centrifugation and freeze-dried. Polymers were collected according to the method of Example 4.

[0123] In addition, a portion of the cells obtained by centrifugation were dried in a desiccator at 80°C for 48 hours, and the PLA content in the cells was analyzed by gas chromatography. Methyl 4-hydroxybutyrate, PLA homopolymer, and P(3HB-co-3HV) copolymer containing 12 wt% 3HV were used as standards.

[0124] As a result, 3HB and 4HB were not detected, and only PLA was detected. Based on the weight of the dried cells, the PLA content synthesized in *E. coli* was approximately 10%. Gas chromatography analysis of the final polymer confirmed that the obtained polymer was a polylactic acid ester polymer with a PLA content of 99.1 mol%.

[0125] <Example 6> Preparation of Multiple Mutants

[0126] Various PHA synthase mutants were prepared using the primers described below, following the same method disclosed in Example 2 above. The resulting mutants are shown in Tables 4, 5, 6, and 7.

[0127] E130D

[0128] SEQ ID NO:15:5'-atc aac ctc atg acc gat gcg atg gcg ccg acc-3'

[0129] SEQ ID NO:16:5'-ggt cgg cgc cat cgc atc ggt cat gag gtt gat-3'

[0130] S325T

[0131] SEQ ID NO:11:5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC-3'

[0132] SEQ ID NO:12:5'-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG-3'

[0133] S477R

[0134] SEQ ID NO:21:5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc-3'

[0135] SEQ ID NO:22:5'-gat atg ccc gcg gct cga cag cac gaa ttc-3'

[0136] S477H

[0137] SEQ ID NO:23:5'-gaa ttc gtg ctg tcg agc cat ggg cat atc-3'

[0138] SEQ ID NO:24:5'-gat atg ccc atg gct cga cag cac gaa ttc-3'

[0139] S477F

[0140] SEQ ID NO:25:5'-gaa ttc gtg ctg tcg agc ttt ggg cat atc-3'

[0141] SEQ ID NO:26:5'-gat atg ccc aaa gct cga cag cac gaa ttc-3'

[0142] S477Y

[0143] SEQ ID NO:27:5'-gaa ttc gtg ctg tcg agc tat ggg cat atc-3'

[0144] SEQ ID NO:28:5'-gat atg ccc ata gct cga cag cac gaa ttc-3'

[0145] S477G

[0146] SEQ ID NO:29:5'-gaa ttc gtg ctg tcg agc ggc ggg cat atc-3'

[0147] SEQ ID NO:30:5'-gat atg ccc gcc gct cga cag cac gaa ttc-3'

[0148] Q481K

[0149] SEQ ID NO:31:5'-ggg cat atc aaa agc atc ctg aac ccg c-3'

[0150] SEQ ID NO:32:5'-gcg ggt tca gga tgc ttt tga tat gcc c-3'

[0151] Q481M

[0152] SEQ ID NO:33:5'-ggg cat atc atg agc atc ctg aac ccg c-3'

[0153] SEQ ID NO:34:5'-gcg ggt tca gga tgc tca tga tat gcc c-3'

[0154] Q481R

[0155] SEQ ID NO:35:5'-ggg cat atc cgc agc atc ctg aac ccg c-3'

[0156] SEQ ID NO:36:5'-gcg ggt tca gga tgc tgc gga tat gcc c-3'

[0157] Table 4

[0158]

[0159]

[0160] Table 5

[0161]

[0162]

[0163] Table 6

[0164]

[0165] Table 7

[0166]

[0167] <Example 7> Synthesis of P(3HB-co-LA) using multiple mutants

[0168] Recombinant *Escherichia coli* capable of expressing a PHA synthase mutant and propionyl-CoA transferase derived from *Pseudomonas* 6-19 was constructed using the same method described in Example 3 above, and P(3HB-co-LA) was prepared using this recombinant *E. coli* via the same method described in Example 4 above. The results are shown in Tables 8, 9, and 10.

[0169] Table 8

[0170]

[0171] Table 9

[0172]

[0173] Table 10

[0174]

[0175] As shown in Tables 8, 9 and 10, the PHA synthase mutant of the present invention can effectively use lactyl-CoA as a substrate to prepare lactate copolymers.

[0176] [Industrial Applicability]

[0177] As shown above, the polyhydroxyalkanoate synthase mutant of the present invention, derived from Pseudomonas 6-19, can efficiently prepare lactate polymers and / or copolymers by using lactyl-CoA, which is difficult to use as a substrate with conventional polyhydroxyalkanoate synthases.

Claims

1. A process for preparing a lactate homopolymer or lactate copolymer, wherein, The method comprises culturing a microorganism comprising a gene encoding propionyl-CoA transferase (pct) and a gene encoding a polyhydroxyalkanoate synthase mutant using lactyl-CoA as a substrate, wherein the polyhydroxyalkanoate synthase mutant is derived from Pseudomonas sp. 6-19 and is the amino acid sequence of SEQ. ID No: 10, wherein the amino acid sequence has any one of the following mutations: b) E130D and Q481K; d) E130D and Q481M; e) E130D and Q481R; f) E130D, S325T and Q481M; g) E130D, S325T and Q481K; h) E130D, S477R and Q481K; i) E130D, S477R and Q481M; j) E130D, S477R and Q481R; k) E130D, S477H and Q481K; l) E130D, S477H and Q481M; m) E130D, S477H and Q481R; n) E130D, S477F and Q481K; o) E130D, S477F and Q481M; p) E130D, S477F and Q481R; q) E130D, S477Y and Q481K; r) E130D, S477Y and Q481M; s) E130D, S477Y and Q481R; t) E130D, S325T, S477R and Q481M; u) E130D, S325T, S477R and Q481K; v) E130D, S325T, S477F and Q481M; or w) E130D, S325T, S477G and Q481M.

2. The method of claim 1, wherein, The culturing is in a culture medium containing a hydroxyalkanoate, and the copolymer is a copolymer containing hydroxyalkanoate monomer units and lactate monomer units.

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