Mutant of propionyl-CoA transferase from Clostridium propionicum and preparing method for PLA or PLA copolymer using the same

Abstract

Provided is a mutant of propionyl-CoA transferase from Clostridium propionicum that can convert lactate into lactyl-CoA with high efficiency in a method of preparing a polylactate (PLA) or PLA copolymer using microorganisms. Unlike conventional propionyl-CoA transferase which is weakly expressed in E. coli, when a mutant of propiony-CoA transferase from Clostridium propionicum is introduced into recombinant E. coli, lactyl-CoA can be supplied very smoothly, thereby enabling highly efficient preparation of polylactate (PLA) and PLA copolymer.

Claims

1. An isolated mutant gene encoding an isolated mutant of the propionyl-CoA transferase supplying lactyl-CoA, which has a gene sequence of SET ID NO: 3 in which A1200G is mutated, and wherein the gene sequence is selected from the group consisting of: a) a gene sequence of SEQ ID NO: 3, in which A1200G is mutated and one mutation of the nucleic acid is further introduced to cause mutation of Gly335Asp; b) a gene sequence of SEQ ID NO: 3, in which A1200G is mutated and one mutation of the nucleic acid is further introduced to cause mutation of Ala243Thr; and c) a gene sequence of SEQ ID NO: 3, in which A1200G is mutated and one mutation of the nucleic acid is further introduced to cause mutation of Asp257Asn.

2. A recombinant vector for synthesizing a polylactate (PLA) or PLA copolymer, containing the isolated mutant gene according to claim 1.

3. The recombinant vector according to claim 2, further containing a polyhydroxyalkanoate (PHA) synthase gene (phaC1Ps6-19300) of SEQ ID NO: 4, which is capable of synthesizing the PLA or PLA copolymer using lactyl-CoA as a substrate.

4. A microorganism transformed with the recombinant vector according to claim 2.

5. A method of preparing a polylactate (PLA) or PLA copolymer, comprising culturing the microorganism according to claim 4.

6. The method according to claim 5, wherein the culturing is performed in an environment containing hydroxyalkanoate.

7. The method according to claim 6, wherein the hydroxyalkanoate is at least one selected from the group consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, medium chain-length (D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms, 2-hydroxypropionic 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-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5-hydroxyvaleric acid, 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-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7-octenoic 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-cistetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric 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-octenoic acid, malic acid, 3-hydroxysuccinic acid-methylester, 3-hydroxyadipinic acid-methylester, 3-hydroxysuberic acid-methylester, 3-hydroxyazelaic acid-methylester, 3-hydroxysebacic acid-methylester, 3-hydroxysuberic acid-ethylester, 3-hydroxysebacic acidethylester, 3-hydroxypimelic acid-propylester, 3-hydroxysebacic acid-benzylester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-3-hydroxybutyric acid, paracyanophenoxy-3-hydroxyvaleric acid, para-cyanophenoxy-3-hydroxyhexanoic acid, paranitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric acid, 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-heptenoic acid.

8. The method according to claim 7, wherein the hydroxyalkanoate is at least one selected from the group consisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 2-hydroxypropionic acid, 3-hydroxypropionic acid, medium chain-length (D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms, 3-hydroxyvalerate, 4-hydroxyvaleric acid and 5-hydroxyvaleric acid.

9. The method according to claim 8, wherein the hydroxyalkanoate is 3-hydroxybutyrate.

10. A microorganism transformed with the recombinant vector according to claim 3.

11. A method of preparing a polylactate (PLA) or PLA copolymer, comprising culturing the microorganism according to claim 10.

12. The method according to claim 11, wherein the culturing is performed in an environment containing hydroxyalkanoate.

13. The method according to claim 12, wherein the hydroxyalkanoate is at least one selected from the group consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, medium chain-length (D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms, 2-hydroxypropionic 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-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5-hydroxyvaleric acid, 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-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7-octenoic 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-cistetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric 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-octenoic acid, malic acid, 3-hydroxysuccinic acid-methylester, 3-hydroxyadipinic acid-methylester, 3-hydroxysuberic acid-methylester, 3-hydroxyazelaic acid-methylester, 3-hydroxysebacic acid-methylester, 3-hydroxysuberic acid-ethylester, 3-hydroxysebacic acidethylester, 3-hydroxypimelic acid-propylester, 3-hydroxysebacic acid-benzylester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-3-hydroxybutyric acid, paracyanophenoxy-3-hydroxyvaleric acid, para-cyanophenoxy-3-hydroxyhexanoic acid, paranitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric acid, 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-heptenoic acid.

14. The method according to claim 13, wherein the hydroxyalkanoate is at least one selected from the group consisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 2-hydroxypropionic acid, 3-hydroxypropionic acid, medium chain-length (D)-3-hydroxycarboxylic acid with 6 to 14 carbon atoms, 3-hydroxyvalerate, 4-hydroxyvaleric acid and 5-hydroxyvaleric acid.

15. The method according to claim 14, wherein the hydroxyalkanoate is 3-hydroxybutyrate.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram of a intracellular synthesis pathway of lactate copolymer (poly(3HB-co-lactate)) using glucose, 3HB and lactate.

(2) FIG. 2 is a schematic diagram of a process of preparing a recombinant expression vector containing polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 6-19, and a mutant gene of propionyl-CoA transferase from Clostridium propionicum, according to an example of the present invention.

MODE FOR INVENTION

(3) Hereinafter, the present invention will be described in detail through examples. It is obvious to one skilled in the art that the examples is provided only to explain the present invention in detail, the present invention is not limited to the examples

(4) According to a previous invention of the present inventors disclosed in Korean Patent Application No 10-2006-0116234, in order to provide lactyl-CoA that is a monomer required for synthesis of polylactate (PLA) and PLA copolymer, an operon-type type constant expression system in which propionyl-CoA transferase from Clostridium propionicum (CP-PCT) and polyhydroxyalkanoate (PHA) synthase are expressed together was constructed as will now be described in more detail.

Example 1-1: Cloning of a PHA Synthase Gene from Pseudomonas sp. 6-19 and Preparation of Expression Vector

(5) In order to separate PHA synthase ((phaC1.sub.Ps6-19) gene from Pseudomonas sp. 6-19(KCTC 11027BP) used in the present invention, the total DNA of Pseudomonas sp. 6-19 was extracted, primers having base sequences of SEQ ID NOs: 5 and 6 were prepared based on a sequence of the phaC1.sub.Ps6-19 gene (Ae-jin Song, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004), and polymerase chain reaction (PCR) was performed to obtain the phaC1.sub.Ps6-19 gene.

(6) TABLE-US-00001 SEQIDNO:5: 5-GAGAGACAATCAAATCATGAGTAACAAGAGTAA CG-3 SEQIDNO:6: 5-CACTCATGCAAGCGTCACCGTTCGTGCACGTAC-3

(7) As a result of performing Agarose gel electrophoresis of the PCR product, a 1.7-kbp gene fragment corresponding to phaC1.sub.Ps6-19 gene was confirmed.

(8) A DNA fragment containing a PHB-producing operon from Ralstorda eutropha H16 was cut with BamHI/EcoRI from a pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347), and inserted into a BamHI/EcoRI recognition site of pBluescript II (Stratagene), thereby preparing a pReCAB recombinant vector.

(9) It is known that the pReCAB vector in which PHA synthase (phaC.sub.RE) and monomer-supplying enzymes (phaA.sub.RE and phaB.sub.RE) are constantly expressed by a PHB operon promoter, is also effectively operated in E. coli. (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347). The pReCAB vector was cut with BstBI/SbfI to remove R. eutropha H16 PHA synthase (phaC.sub.RE), and the phaC1.sub.Ps6-19 gene was inserted into a BstBI/SbfI recognition site, thereby preparing a pPs619C1-ReAB recombinant vector.

(10) In order to produce a phaC1.sub.Ps6-19 synthase gene fragment having only a BstBI/SbfI recognition site on either end, an endogenous BstBI site was removed using site-directed mutagenesis (SDM) without change of amino acids, and an overlapping PCR was performed using primers of SEQ ID NOs: 7 and 8, SEQ ID NOs:9 and 10, and SEQ ID NOs: 11 and 12 in order to add the BstBI/SbfI recognition site.

(11) TABLE-US-00002 SEQIDNO:7: 5-ATGCCCGGAGCCGGTTCGAA-3 SEQIDNO:8: 5-CGTTACTCTTGTTACTCATGATTTGATTGTCTC TC-3 SEQIDNO:9: 5-GAGAGACAATCAAATCATGAGTAACAAGAGTAA CG-3 SEQIDNO:10: 5-CACTCATGCAAGCGTCACCGTTCGTGCACGTAC-3 SEQIDNO:11: 5-GTACGTGCACGAACGGTGACGCTTGCATGAGTG-3 SEQIDNO:12: 5-AACGGGAGGGAACCTGCAGG-3

(12) The base sequence of the phaC1.sub.Ps6-19 gene of the prepared pPs619C1-ReAB recombinant vector was confirmed by sequencing and represented by SEQ ID NO: 13, and the amino acid sequence coded thereby was represented by SEQ ID NO: 14.

(13) The gene sequence similarity analysis results show that the phaC1.sub.Ps6-19 gene has a homology of 84.3% with phaC1 from Pseudomonas sp. strain 61-3 (Matsusaki et al., J. Bacteriol., 180:6459, 1998) and an amino-acid sequence homology of 88.9%. Thus, it was confirmed that the two synthases were very similar enzymes. As a result, it was confirmed that the phaC1.sub.Ps6-19 synthase obtained according to the invention was a Type II PHA synthase.

Example 1-2: Preparation of a Substrate-Specific Mutant of a PHA Synthase from Pseudomonas sp. 6-19

(14) Among various kinds of PHA synthases, a Type II PHA synthase is known as a medium-chain-length PHA (MCL-PHA) synthase for polymerizing a substrate having relatively many carbon atoms, and the MCL-PHA synthase is expected to be very applicable to production of a PLA copolymer. Although phaC1 synthase from Pseudomonas sp. 61-3, which has a high homology with the phaC1.sub.Ps6-19 synthase obtained according to the present invention, is the Type II synthase, it was reported that the phaC1 synthase had a relatively wide range of substrate specificity (Matsusaki et al., J. Bacteriol., 180:6459, 1998), and results of research in a mutant suitable for production of short-chain-length PHA (SCL-PHA) were reported (Takase et al., Biomacromolecules, 5:480, 2004). Based on the above, the present inventors found three amino-acid sites affecting SCL activation via amino-acid sequence arrangement analysis, and prepared mutants of phaC1.sub.Ps6-19 synthase by an SDM method using primers of SEQ ID NOs: 15 to 20 as shown in Table 1.

(15) TABLE-US-00003 TABLE1 Nucleicacid Aminoacid Recombinantvector substitution substitution Primer pPs619C1200-ReAB AGC.fwdarw. ACC S325T SEQIDNOs:15/16 CAG.fwdarw. ATG Q481M SEQIDNOs:17/18 GAA.fwdarw. GAT E130D SEQIDNOs:19/20 AGC.fwdarw. ACC S325T SEQIDNOs:15/16 CAG.fwdarw. ATG Q481M SEQIDNOs:17/18

(16) TABLE-US-00004 SEQIDNO:15: 5-CTGACCTTGCTGGTGACCGTGCTTGATACCACC-3 SEQIDNO:16: 5-GGTGGTATCAAGCACGGTCACCAGCAAGGTCAG-3 SEQIDNO:17: 5-CGAGCAGCGGGCATATCATGAGCATCCTGAACC CGC-3 SEQIDNO:18: 5-GCGGGTTCAGGATGCTCATGATATGCCCGCTGC TCG-3 SEQIDNO:19: 5-ATCAACCTCATGACCGATGCGATGGCGCCGACC-3 SEQIDNO:20: 5-GGTCGGCGCCATCGCATCGGTCATGAGGTTGAT-3

Example 1-3: Preparation and Screening of a Library of a Mutant of Propionyl-CoA Transferase from Clostridium propionicum

(17) In the present example, in order to provide lactyl-CoA that is a monomer required for synthesis of PLA and PLA copolymer, propionyl-CoA transferase from Clostridium propionicum (CP-PCT) was used and its sequence was represented by SEQ ID NO: 3. A fragment obtained by performing PCR on chromosomal DNA of Clostridium propionicum using primers of SEQ ID NOs: 21 and 22 was used as CP-PCT. In this case, an NdeI site existing in wild-type CP-PCT was removed using SDM to facilitate cloning.

(18) TABLE-US-00005 SEQIDNO:21: 5-GGAATTCATGAGAAAGGTTCCCATTATTACCGC AGATGA-3 SEQIDNO:22: 5-GCTCTAGATTAGGACTTCATTTCCTTCAGACC CATTAAGCCTTCTG-3

(19) Also, overlapping PCR was performed using a primer of SEQ ID NOs: 23 and 24 in order to add a SbfI/NdeI recognition site.

(20) TABLE-US-00006 SEQIDNO:23: 5-aggcctgcaggcggataacaatttcacacagg-3 SEQIDNO:24: 5-gcccatatgtctagattaggacttcatttcc-3

(21) A pPs619C1300-ReAB vector containing phaC1.sub.Ps6-19300, which is an SCL mutant of phaC1.sub.Ps6-19 synthase, was cut with SbfI/NdeI to remove a monomer-supplying enzyme (phaA.sub.RE and phaB.sub.RE) from R. eutropha H16, and the PCR-cloned CP-PCT gene was inserted into the SbfI/NdeI recognition site, thereby preparing a pPs619C1300-CPPCT recombinant vector.

Example 2: Preparation and Screening of a Library of a Mutant of Propionyl-CoA Transferase from Clostridium propionicum

(22) As is known, when CP-PCT is highly expressed in E. coli, it causes serious metabolic disorder and exhibits toxicity. In general, recombinant E. coli were dead at the same time with addition of an inducer in an isopropyl--D-thio-galactoside (IPTG)-inducible protein production system using a tac promoter or T7 promoter, which is widely used to express recombinant proteins. For this reason, synthesis of PLA and PLA copolymer was performed using a constitutive expression system which weakly but continuously expressed genes with growth of microorganisms. In order to introduce random mutation into CP-PCT, pPs619C1300-CPPCT disclosed in Korean Patent Application No. 10-2006-0116234 was used as a template, and error-prone PCR was performed using primers of SEQ ID NOs: 1 and 2 under conditions of addition of Mn.sup.2+ and the difference of concentration between dNTPs (refer to FIG. 2).

(23) TABLE-US-00007 SEQIDNO:1: 5-cgccggcaggcctgcagg-3 SEQIDNO:2: 5-ggcaggtcagcccatatgtc-3

(24) Thereafter, in order to amplify a PCR fragment including random mutation, PCR was performed under common conditions using primers of SEQ ID NOs: 1 and 2. A pPs619C1300-CPPCT vector containing phaC1.sub.Ps6-19300, which is an SCL mutant of phaC1.sub.Ps6-19 synthase, was cut with SbfI/NdeI to remove wild-type CP-PCT, and the amplified mutant PCR fragment was inserted into the SbfI/NdeI recognition site to produce a ligation mixture. The ligation mixture was introduced into E. coli JM109, thereby preparing a CP-PCT library with a scale of about 10^5 (refer to FIG. 2). The prepared CP-PCT library was grown for 3 days in a polymer detection medium (a Luria Bertani (LB) agar, glucose 20 g/L, 3HB 1 g/L, Nile red 0.5 g/ml) and screened to confirm whether polymer was generated, thereby primarily selecting 80 candidates. The candidates were grown for 4 days in a liquid medium (an LB agar, glucose 20 g/L, 3HB 1 g/L, ampicillin 100 mg/L, 37 C.) under polymer generation conditions and analyzed using florescence activated cell sorting (FACS), thereby selecting final two samples. Also, a recombinant expression vector containing the mutant was retrieved from E. coli and introduced into E. coli JM109 again to confirm polymer yield. Thus, it was confirmed that the E. coli transformed with a recombinant vector having a mutant of CP-PCT exhibited better performance than that having wild-type CP-PCT.

Example 3: Preparation of a PLA Copolymer Using a Mutant of Propionyl-CoA Transferase from Clostridium propionicum

(25) In order to quantitatively analyze activation of mutants finally selected in Example 2, E. coli JM109 transformed with the recombinant expression vector containing the mutants as shown in FIG. 2 was cultured for 4 days at a temperature of in a flask having an LB medium containing glucose (20 g/L) and 3HB (2 g/L). The cultured cell pellet was retrieved by centrifugation and dried for 24 hours in a dryer at a temperature of 100. Thereafter, gas chromatography was performed to estimate the polymer contents synthesized in cells as shown in Table 2.

(26) TABLE-US-00008 TABLE 2 PLA content PHB content Name of Strain (w/w %) (w/w %) pPs619C1300-CPPCT/JM109 0.86% 5.85% CP-PCT mutant 512/JM109 2.19% 12.82% CP-PCT mutant 522/JM109 7.49% 35.59%

(27) As a result of the gas chromatographic analysis, it can be seen that the recombinant expression vector containing the mutant of CP-PCT prepared according to the present invention had about two to eight times the PLA-copolymer synthetic activity of the pPs619C1300-CPPCT vector containing wild-type CP-PCT. This is because the mutant of the CP-PCT supplies polymer synthesis monomers (i.e., lactyl-CoA and 3HB-CoA) more efficiently than the wild-type CP-PCT.

(28) In order to find out the mutant positions of the prepared CP-PCT mutants, gene sequencing was performed with respect to the CP-PCT mutants, and the results were shown in Table 3.

(29) TABLE-US-00009 TABLE 3 Recombinant vector Nucleic acid substitution CP-PCT mutant 512 A1200G CP-PCT mutant 522 T78C, T669C, A1125G, T1158C

(30) As a result of the gene sequencing of the CP-PCT mutants, one nucleic-acid substitution occurred in the mutant 512, while four nucleic-acid substitutions occurred in the mutant 522. However, it was confirmed that all the nucleic-acid substitutions were silent mutations that cause no amino-acid substitution. That is, it was assumed that an improvement in monomer-supplying capability of the CP-PCT mutants prepared according to the present invention resulted not from an increase in activity due to amino-acid substitution of an enzyme but from a variation in the enzyme expression in E. coli. Thus, the codon usages of gene sequences of the wild-type CP-PCT and CP-PCT mutants prepared according to the present invention in typical E. coli were analyzed as shown in Table 4.

(31) TABLE-US-00010 TABLE4 d 78 669 1125 1158 1200 Wild-typeCP-PCR GGT(Gly) GGT(Gly) AAA(Lys) CGT(Arg) ACA(Thr) 24.7 24.7 33.6 20.9 7.1 CP-PCTmutant512 GGT(Gly) GGT(Gly) AAA(Lys) CGT(Arg) ACA(Thr) 24.7 24.7 33.6 20.9 14.4 CP-PCTmutant522 GGC(Gly) GGC(Gly) AAG(Lys) CGC(Arg) ACA(Thr) 29.6 29.6 10.3 22.0 7.1

(32) As shown in Table 4, all the nucleic-acid substitutions except A1125G existing in the mutant 522 were advantageous for the codon usage of E. coli. That is, due to the nucleic-acid substitution being advantageous for the codon usage of the E. coli, the CP-PCT mutants prepared according to the present invention increased the expression of an activated enzyme and thus exhibited higher monomer-supplying capabilities required for production of PLA copolymers.

Example 4: Preparation of a PLA Copolymer Using a Mutant of Propionyl-CoA Transferase from Clostridium propionicum

(33) Random mutagenesis was performed on the mutants (512 and 522) finally selected in Example 2, in the same manner as described in Example 2, thereby obtaining the following CP-PCT mutants 531-537 and 540.

(34) In order to quantitatively analyze the CP-PCT mutants, E. coli JM109 transformed with the recombinant expression vector containing the CP-PCT mutants 531-537 and 540 was cultured for 4 days at a temperature of 30 C. in a flask having a P-rich methyl red (MR) medium containing glucose (20 g/L) and 3HB (2 g/L). The cultured cell pellet was retrieved by centrifugation and dried for 24 hours in a dryer maintained at a temperature of 100 C. Thereafter, gas chromatography was performed to estimate the contents of polymers synthesized in cells. The results of gene sequencing with respect to the CP-PCT mutants performed to find out the mutation positions of the CP-PCT mutants are shown in Table 5, and the contents of the polymers synthesized in the cells are shown in Table 6.

(35) In the above-described experiment, P-rich MR was formed of 22 g of KH.sub.2PO.sub.4; 3 g of (NH.sub.4).sub.2HPO.sub.4; 0.8 g of Citrate; 0.7 g of MgSO.sub.4.7H.sub.2O; and 5 mL of a trace metal solution per liter, and the trace metal solution was formed of 10 g of FeSO.sub.4.7H.sub.2O; 2.25 g of ZnSO.sub.4.7H.sub.2O; 1 g of CuSO.sub.4.5H.sub.2O; 0.5 g of MnSO.sub.4.5H.sub.2O; 2 g of CaCl.sub.2.2H.sub.2O; 0.23 g of Na.sub.2B.sub.4O.sub.7.7H.sub.2O; 0.1 g of (NH.sub.4).sub.6Mo.sub.7O.sub.24; and 10 mL of HCl 35% per liter.

(36) TABLE-US-00011 TABLE 5 Mutations Silent Mutations CpPct512 A1200G CpPct522 T78C, T669C, A1125G, T1158C CpPct531 Gly335Asp A1200G CpPct532 Ala243Thr A1200G CpPct533 Asp65Gly T669C, A1125G, T1158C CpPct534 Asp257Asn A1200G CpPct535 Asp65Asn T669C, A1125G, T1158C CpPct537 Thr199Ile T669C, A1125G, T1158C CpPc1540 Val193Ala T78C, T669C, A1125G, T1158C

(37) TABLE-US-00012 TABLE 6 Polymer content Lac 3HB Name (%) mol % mol % PCT (wild control) 24.9 38.0 62.0 531 26.5 56.5 43.5 532 23.5 54.5 45.5 533 25.2 63.8 36.2 534 23.9 58.0 42.0 535 30.7 59.2 40.8 537 33.3 52.4 47.6 540 23.7 21.3 78.7

(38) As shown in Table 6, the CP-PCT mutants prepared according to the present invention remarkably increased lactate mole % compared to wild-type CP-PCT. Also, it was confirmed that the recombinant expression vector containing the mutants 531, 533, 535, 537, and 540 exhibited better PLA-copolymer synthetic activity than the vector containing wild-type CP-PCT.

(39) While the invention has been shown and described with reference to certain examples thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.