# Patent

## Title
Recombinant microorganism having ability to produce [lactate-co-glycolate] copolymer from glucose, and method for preparing [lactate-co-glycolate] copolymer using same

## Abstract

There is provided a recombinant microorganism having producibility of poly(lactate-co-glycolate) from glucose, and more particularly, a recombinant microorganism having producibility of poly(lactate-co-glycolate) without adding an exogenous glycolate precursor, and a method of preparing [poly(preparing lactate-co-glycolate)] using the same. According to the present invention, the poly(lactate-co-glycolate) in which the concentration of the glycolate fraction is high may be prepared at a high concentration without supplying exogenous glyoxylate. Therefore, the present invention may be effectively used for treatment.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national phase under 35 U.S.C. §371 of International Patent Application No. PCT/KR12/01754 filed Mar. 9, 2012, which in turn claims priority of Korean Patent Application No. 10-2011-0022020 filed Mar. 11, 2011. The disclosures of such international patent application and Korean priority patent application are hereby incorporated herein by reference in their respective entireties, for all purposes.
TECHNICAL FIELD
The present invention relates to a recombinant microorganism having producibility of poly(lactate-co-glycolate) from glucose, and more particularly, to a recombinant microorganism having producibility of poly(lactate-co-glycolate) without adding an exogenous glycolate precursor, and a method of preparing poly(preparing lactate-co-glycolate) using the same.
BACKGROUND ART
Poly(lactate-co-glycolate) (PLGA), which is a representative biodegradable polymer derived from lactate and glycolate, is a polymer having high applicability to a general purpose polymer or medical polymer. Currently, the PLGA may be prepared by a direct polymerization reaction of lactate and glycolate, but PLGA having a low molecular weight (1000 to 5000 daltons) is mainly prepared in this reaction. PLGA having a high molecular weight of 100,000 daltons or more may be synthesized by a ring opening condensation reaction of lactide and glycolide. The lactide and glycolide, which are cyclic diesters of lactate and glycolate, respectively, are formed by pyrolysis of a lactate oligomer and a glycolate oligomer, respectively.
In the ring opening condensation reaction, a catalyst such as tin(II) 2-ethylhexanoate, tin(II) alkoxide, aluminum isopropoxide, or the like, should be used. As a method of preparing the PLGA having a high molecular weight, there is a method of polymerizing a polymer having a relatively high molecular weight from a polymer obtained by direct polymerization and having a low molecular weight using a chain coupling agent, but in this method, since the chain coupling agent is used, a process may be complicated due to addition of an organic solvent or the chain coupling agent, and it may be difficult to remove this organic solvent or chain coupling agent.
Currently, in a commercialized process for producing the PLGA having a high molecular weight, a method of converting lactate and glycolate into lactide and glycolide, respectively, and then synthesizing the PLGA through the ring opening condensation reaction of the lactide and glycolide has been used.
Meanwhile, poly(hydroxyalkanoate) (PHA) is a polyester accumulated by microorganism as an energy or carbon source storage material in the microorganism when the carbon source excessively exists but other nutrients such as phosphorus, nitrogen, magnesium, oxygen, and the like are insufficient. Since the PHA has complete biodegradability while having physical properties similar to those of the existing synthetic polymers derived from petroleum, the PHA has been recognized as a material replacing the existing synthetic plastic material.
The existing known PHA may be representatively divided into a short-chain-length PHA (SCL-PHA) having a short carbon chain and a medium-chain-length PHA (MCL-PHA) having a long carbon chain. Gene for synthesizing the PHA was cloned from Ralstonia eutropha, Pseudomonas, or the like, and PHA composed of various monomers was synthesized by recombinant microorganism (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; U.S. Pat. No. 6,143,952; WO 98/54329; WO 99/61624).
Since glycolic acid is the simplest hydrocarboxylic acid, there has been an attempt to insert glycolic acid into the PHA polymer using a PHA synthase enzyme of Ralstonia europha and a glycolyl-CoA produced from beta oxidation pathway as a substrate.
The present inventors confirmed that in the case of culturing recombinant E. coli transfected with propionate CoA-transferase gene (Pct) derived from Clostridium propionicum, which is gene coding an enzyme converting lactate and glycolate into lactyl-CoA and glycolyl-CoA, respectively, and poly(hydroxyalkanoate) (PHA) synthase gene using lactyl-CoA and glycolyl-CoA as substrates in a production medium containing glucose and glycolate or glucose, glycolate, and hydroxyalkanoate, poly(lactate-co-glycolate) and poly(lactate-co-glycolate-co-hydroxyalkanoate) were produced (Korean Patent Application No. 10-2009-0030133).
However, the method of producing PLGA using the recombinant E. coli has a disadvantage in that glucose and glycolate corresponding to precursors of the monomer should be individually added.
Therefore, the present inventors have tried to develop recombinant E. coli capable of producing PLGA in which a content of a glycolate fraction is high without adding exogenous glycolate, and confirmed that in the case of expressing glycerate dehydrogenase of P. multocida or E. coli in E. coli transfected with propionate CoA-transferase of Clostridium propionicum and PHA synthase of Pseudomonas sp. 6-19, deleting isocitrate lyase regulator gene (iclR) and aceB (malate synthase) gene, and amplifying isocitrate lyase gene (aced), even though glycolate is not added, PLGA in which the content of the glycolate fraction is high may be produced at a high concentration using only glucose, thereby completing the present invention.
SUMMARY
An object of the present invention is to provide a recombinant microorganism capable of producing PLGA at a high concentration without adding exogenous glycolate.
Another object of the present invention is to provide a method of preparing PLGA using a recombinant microorganism capable of producing PLGA at a high concentration without adding exogenous glycolate.
According to an aspect of the present invention, there is provided a mutant transfected with a gene coding poly(hydroxyalkanoate) (PHA) synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26).
According to another aspect of the present invention, there is provided a mutant transfected so that a gene coding PHA synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted, a gene (iclR) coding isocitrate lyase regulator or a aceB (malate synthase) gene is deleted.
According to another aspect of the present invention, there is provided a method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing a mutant transfected with a gene coding poly(hydroxyalkanoate) (PHA) synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) in a glucose-containing medium to produce PLGA; and obtaining the produced PLGA.
According to another aspect of the present invention, there is provided a method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing a mutant transfected so that a gene coding PHA synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted, a gene (iclR) coding isocitrate lyase regulator or aceB (malate synthase) gene is deleted, and a gene (aceA) coding isocitrate lyase is amplified in a glucose-containing medium to produce PLGA; and obtaining the produced PLGA.
Other features and embodiments of the present invention will become obvious from the following detailed description and the accompanying claims.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A shows Chemical Formulas of poly(lactate-co-glycolate) (PLGA), poly-gamma-glutamic acid (PGA), and polylactic acid (PLA) prepared in the present invention, and FIG. 1B shows a PLGA production pathway by metabolic engineering performed in the present invention.
FIG. 2 shows a map of genes associated with production of PLGA in chromosome of E. coli XL1-Blue.
FIG. 3 shows a map of genes associated with production of PLGA in chromosome of recombinant E. coli JLXF5 constructed in the present invention.
FIG. 4 shows a map of genes associated with production of PLGA in chromosome of recombinant E. coli JLXF8 constructed in the present invention.
FIG. 5A-C show NMR results of PLGA which is produced by recombinant E. coli constructed in the present invention.



DETAILED DESCRIPTION OF THE EMBODIMENTS
Unless otherwise defined herein, technical and scientific terms used in the present specification have the same meanings as those understood by specialists in the skilled art to which the present invention pertains. Generally, nomenclature used in the present specification is well known and commonly used in the art.
In one general aspect, the present invention relates to a recombinant microorganism transfected with a gene coding poly(hydroxyalkanoate) (PHA) synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) and having producibility of poly(lactate-co-glycolate).
The propionate CoA-transferase may be propionate CoA-transferase of C. propionicum or Pct540, which is a mutant enzyme of propionate CoA-transferase of C. propionicum, and poly(hydroxyalkanoate) (PHA) synthase may be PHA synthase of Pseudomonas sp. 6-19 or a mutant enzyme of PHA synthase of Pseudomonas sp. 6-19.
The gene coding the mutant enzyme of the PHA synthase may have a base sequence corresponding to an amino acid sequence including at least one mutation selected from a group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in an amino acid sequence of SEQ ID No: 1. In addition, the gene coding the mutant enzyme of the PHA synthase may have a base sequence corresponding to an amino acid sequence selected from a group consisting of an amino acid sequence C1335 in which E130D, S325T, L412M, S477G, and Q481M in the amino acid sequence of SEQ ID No: 1 are mutated; an amino acid sequence C1310 in which E130D, S477F, and Q481K in the amino acid sequence of SEQ ID No: 1 are mutated; and an amino acid sequence C1312 in which E130D, S477F, and Q481R in the amino acid sequence of SEQ ID No: 1 are mutated.
The gene coding glycerate dehydrogenase (EC 1.1.1.26) may be derived from Pasteurella multocida or E. coli and have a base sequence of SEQ ID No: 2.
In another aspect, the present invention relates to a recombinant microorganism transfected so that a gene coding PHA synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted, a gene (iclR) coding isocitrate lyase regulator or aceB (malate synthase) gene is deleted, and a gene (aceA) coding isocitrate lyase is amplified, and having producibility of poly(lactate-co-glycolate).
In the present invention, the term “deletion” means that genes are modulated so as to allow a protein coded by the gene not to perform its original function through substitution, removal, and mutation.
In the present invention, both of the gene (iclR) coding isocitrate lyase regulator and aceB (malate synthase) genes may be deleted.
In the present invention, in the recombinant microorganism, the gene (aceA) coding isocitrate lyase may be additionally amplified.
In the present invention, a parent strain of the mutant may be selected from a group consisting of E. coli XL1-Blue, E. coli JLX5, E. coli JLX6, E. coli JLX7, E. coli JLX8, E. coli JLX10, E. coli JLX11, E. coli JLX12, and E. coli JLX13, and features of the parent strain were shown in Table 1.
In another aspect, the present invention relates to a method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing a recombinant microorganism transfected with a gene coding poly(hydroxyalkanoate) (PHA) synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) in a glucose-containing medium to produce PLGA; and obtaining the produced PLGA.
In the present invention, an E. coli strain producing the PLGA, which is a polymer that is not produced in a natural state, was developed, and a production amount of glycolate, which is a monomer of the PLGA, may be further increased by modulating a metabolic pathway of E. coli. 
In one aspect, a recombinant E. coli JLXF5 strain expressing Pct540Cp, PhaC1437Ps6-19, and glycerate dehydrogenase of P. multocida produces 33.6 weight % of P(35.2 mol % LA-co-64.8 mol % GA). This result is a first study in which the poly(lactate-co-glycolate) is produced from glucose in metabolically engineered recombinant E. coli. 
In another aspect, the present invention relates to a method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing a recombinant microorganism in which a gene coding PHA synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted, a gene (iclR) coding isocitrate lyase regulator or aceB (malate synthase) gene is deleted in a glucose-containing medium to produce PLGA; and obtaining the produced PLGA.
EXAMPLE
Hereinafter, the present invention will be described in detail through the Examples. However, these Examples are only to illustrate the present invention, and those skilled in the art will appreciate that these Examples are not to be construed as limiting a scope of the present invention.
Example 1
Used Strain and Analysis Method
Strains, plasmids, and primers used in the following Examples were shown in Table 1.
In the following Example, a MR medium used to produce a PLGA in a recombinant E. coli may contain 6.67 g of KH2PO4, 4 g of (NH4)2HPO4, 0.8 g of MgSO4H2O, 0.8 g of citric acid, and 5 ml of a trace metal solution per 1 L, wherein the trace metal solution contains 0.5M HCl: 10 g of FeSO4.H2O, 2 g of CaCl2, 2.2 g of ZnSO4.H2O, 0.5 g of MnSO4.H2O, 1 g of CuSO4.H2O, 0.1 g of (NH4)6Mo7O24.H2O, and 0.02 of Na2B4O7.10H2O, Carbon source, and MgSO4.H2O per 1 L.
In culturing each strain, a seed strain was shaking-cultured in a 25 mL tube in which 10 mL of LB medium was added at 30° C. overnight, and 1 mL of the cultured solution was inoculated into a 250 mL flask charged with 100 mL of the MR medium containing glucose (20 g/L) and shaking-cultured at 30° C. for 72 hours. In the case of synthesizing p(3HB-co-LA-co-GA), 3HB (2 g/L) was added to a culture medium.
In the case of using JLX10 strains (Jung et al., 2010) as a host cell, in order to overcome growth restriction caused by ppc gene deletion, 4 g/L of sodium succinate (Sigma-Aldrich, USA) was added to the culture medium, and in order to express ldhA and acs genes regulated by a trc promoter, when an OD600 value was 0.5, 1 mM IPTG was added. As needed, ampicillin (100 μM/mL), chloramphenicol (34 μg/mL), and thiamine (10 μg/mL) were added to the medium.
Genetic characteristics of the recombinant strains and plasmids used in the present invention were shown in Table 1, and the primers used to construct the recombinant strains and plasmids in the present invention were shown in Table 2.
In constructing the recombinant strain used in the present invention, as a method of deleting iclR and aceB genes in chromosome and replacing a promoter of aceA gene with a trc promoter, a one-step inactivation method using the primer shown in Table 2 was used (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA. 6; 97:6640, 2000).









TABLE 1





Strain,




Plasmid or

Reference or


Primer
characteristica
rececing area







Strain




XL1-Blue
recA1 endA1 gyrA96 thi-1 hsdR17
Stratageneb



supE44 relA1 lac [F′ proAB lacIqZΔM15




Tn10 (TetR)]



 


JLX10
XL1-Blue ΔackA PldhA::Ptrc Δppc
Present invention



Pacs::Ptrc ΔadhE



 


JLX11
JLX10 ΔiclR
Present invention


 


JLX12
JLX10 ΔaceB PaceA::Ptrc
Present invention


 


JLX13
JLX10 ΔiclR ΔaceB PaceA::Ptrc
Present invention


 


JLXF5
XB-F ΔiacI ΔpflB ΔfrdABCD ΔadhE
Present invention



PldhA::Ptrc Pacs::Ptrc



 


JLXF6
JLXF5 ΔiclR
Present invention


 


JLXF7
JLXF5 ΔaceB PaceA::Ptrc
Present invention


 


JLXF8
JLXF5 ΔiclR ΔaceB PaceA::Ptrc
Present invention


 




Present invention


 


Plasmid




pBluescript
ApR, cloning and expression vector
Stratageneb


 


pPs619C1400-
ApR, Promoter of the C. necator PHA
Present invention


CpPCT532
biosynthesis operon, phaC1Ps6-19




variant (phaC1400Ps6-19; E130D, S325T,




S477R, Q481M), pctCp variant




(pct532Cp; A243T, silent mutations:




A1200G), transcriptional terminator




of the C. necator PHA biosynthesis




operon, derivative of pBluescript II




KS(+)



 


pPs619C1310-
APR, Promoter of the C. necator PHA
Korean Patent


CpPCT540
biosynthesis operon, phaC1Ps6-19
Laid-Open



variant (phaC1310Ps6-19; E130D, S477F,
Publication No.



Q481K), pctCp variant (pct540Cp;
2010-0111766



V193A, Silent mutations: T78C,




T669C, A1125G, T1158C),




transcriptional terminator of the C.




necator PHA biosynthesis operon,




derivative of pBluescriptII KS(+)



 


pPs619C1437-
APR, Promoter of the C. necator PHA
Present invention


CpPCT540
biosynthesis operon, phaC1Ps6-19




variant (phaC1337Ps6-19; E130D, S325T,




S477G, Q481K), pctCp variant




(pct540Cp; V193A, Silent mutations:




T78C, T669C, A1125G, T1158C),




transcriptional terminator of the C.




necator PHA biosynthesis operon,




derivative of pBluescriptll KS(+)



 


pTac15k
p15A origin of replication, KmR, tac
Lab stock



promoter



 


pTac15kPmu0559
Derivative of pTac15k, expression of
Present invention



Pasteurella multocida glycerate




dehydrogenase gene under tac




promoter



 


pBBR1MCS2
Broad host range vector; Kmr



 


pZE12-MCS
Expression vector; PLlacO-1 promoter;
EXPRESSYSc



Apr



 


pKE12-MCS
Expression vector; PLlacO-1 promoter,
Present invention



R. eutropha PHA biosynthesis genes




transcription terminator; Apr



 


pZA31-MCS
Expression vector; PLtetO-1 promoter;
EXPRESSYS



Cmr



 


pKA32-MCS
Expression vector; PLlacO-1 promoter,
Present invention



R. eutropha PHA biosynthesis genes




transcription terminator; Cmr



 


pKM22-MCS
Expression vector; PLlacO-1 promoter;
Present invention



Kmr



 


pKM22-YcdW
Derivative of pKM22-MCS, expression
Present invention



of E. coli glycerate dehydrogenase




gene (ycdW) under lacIO promoter



 


Primer




PmuFEcoRI
GTAGAATTCATGTTAAAAATTGTTTTTCTAGATAGT
Present invention


(SEQ ID NO: 3)
ACC



 


PmuRPstI
GTATGCAACTGCAGTTAAGCAATCTGCTCATTTTTT
Present invention


(SEQ ID NO: 4)
AC





aAp: ampicillin; Km: kanamycin; R: resistance.


bStratagene Cloning System, La Jolla, CA, USA.


cwww.expressys.com













TABLE 2





Primer list

















JLX10




K.O.




adhE
FDadhE1
TCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATT



(SEQ ID NO: 5)
ATTAGGTGACACTATAGAACGCG


 



RdadhE1
GATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCGT



(SEQ ID NO: 6)
ATTCGAGCAGATGATTTACTAA


 



FDadhE2
TGATCGGCATTGCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCTA



(SEQ ID NO: 7)
CTTAGTGGATCTGATGGGTACC


 



RdadhE2
GGAAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTTA



(SEQ ID NO: 8)
ACTGATCGGCATTGCCCAGAAG


 


Ptrc




acs
FPacs1
TCACGACAGTAACCGCACCTACACTGTCATGACATTGCTCGCCCCTA



(SEQ ID NO: 9)
TGTGTAACAAATA


 



RPacs1
TGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAAT



(SEQ ID NO: 10)
TGTCAACAGCTAGTGGATCTGATGGGTACC


 



FPacs2
TCACGACAGTAACCGCACCTACACTGTCATGACATTGCTCGCCCCTA



(SEQ ID NO: 11)
TGTGTAACAAATA


 



RPacs2
CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATGGTCTGT



(SEQ ID NO: 12)
TTCCTGTGTGAAATTGTTATCCGCTCACAATTCC


 



FPacs3
CGAATTGCGCCATTGTTGCAATGGCGGTTTTTATTGTTTTTCACGAC



(SEQ ID NO: 13)
AGTAACCGCACCT


 



RPacs3
TTGTTGATACATCGCCTCGTACTGCTGAGGGTTTATCAGGCAACGGT



(SEQ ID NO: 14)
CTGCGATGTTGGCAGGAATGGTG


 


JLXF5




K.O.




pflB
FDpflB1
TTACGGGCCTATAAGCCAGGCGAGATATGATCTATATCAATTTCTCA



(SEQ ID NO: 15)
TCTTAGGTGACACTATAGAACGCG


 



RDpflB1
TTCTTTAGTCAGCGAGTTGAAACGTACTGCGTAGCCAGATACACGGA



(SEQ ID NO: 16)
TGGTAGTGGATCTGATGGGTACC


 



FDpflB2
TATTTGGATAATCAAATATTTACTCCGTATTTGCATAAAAACCATGC



(SEQ ID NO: 17)
GAGTTACGGGCCTATAAGCCAGG


 



RDpflB2
TCTAATTACATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTG



(SEQ ID NO: 18)
CTGTTCTTTAGTCAGCGAGTTGA


 


frdA
FDfrd1
ATTACGTGCTGCAATTGCTGCCGCGCAGGCAAATCCGAATGCAAAAA


BCD
(SEQ ID NO: 19)
TCGTAGGTGACACTATAGAACGCG


 



RDfrd1
GACCGTAGAAAACCCATTTGCCCGCAGGTACGTGGATTTTCAGATCG



(SEQ ID NO: 20)
TGCTAGTGGATCTGATGGGTACC


 



FDfrd2
GTGCAAACCTTTCAAGCCGATCTTGCCATTGTAGGCGCCGGTGGCGC



(SEQ ID NO: 21)
GGGATTACGTGCTGCAATTGCTG


 



RDfrd2
TTAGATTGTAACGACACCAATCAGCGTGACAACTGTCAGGATAGCAG



(SEQ ID NO: 22)
CCAGACCGTAGAAAACCCATTTG


 


adhE
FDadhE1
TCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCAT



(SEQ ID NO: 23)
TATTAGGTGACACTATAGAACGCG


 



RDadhE1
TGATCGGCATTGCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCT



(SEQ ID NO: 24)
ACTTAGTGGATCTGATGGGTACC


 



FDadhE2
GATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCG



(SEQ ID NO: 25)
TATTCGAGCAGATGATTTACTAA


 



RDadhE2
GGAAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTT



(SEQ ID NO: 26)
AACTGATCGGCATTGCCCAGAAG


 


lacI
FDlacI1
TCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGA



(SEQ ID NO: 27)
AAATAGGTGACACTATAGAACGCG


 



RDlacI1
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT



(SEQ ID NO: 28)
GCGTAGTGGATCTGATGGGTACC


 



FDlacI2
GTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTC



(SEQ ID NO: 29)
TTATCAGACCGTTTCCCGCGTGG


 



RDlacI2
CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG



(SEQ ID NO: 30)
TCGTGCCAGCTGCATTAATGAAT


 


Ptrc




ldhA
FPldhA1
CATCTAATGCAATACGTGTCCCGAGCGGTAGCCAGATGCTAGGTGAC



(SEQ ID NO: 31)
ACTATAGAACGCG


 



RPldhA1
TGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAAT



(SEQ ID NO: 32)
TGTCAACAGCTAGTGGATCTGATGGGTACC


 



FPldhA2
GATCGGGAATGATTAAACCTTTACGCGTAATGCGTGGGCTTTCATCT



(SEQ ID NO: 33)
AATGCAATACGTGTC


 



RPldhA2
TCTTGTCGTACTGTTTTGTGCTATAAACGGCGAGTTTCATGGTCTGT



(SEQ ID NO: 34)
TTCCTGTGTGAAATTGTTATCCGCTCACAATTCC


 



FPldhA3
GGTGATATGCGCAAGCTGACAATCTCCCACCAGATAACGGAGATCGG



(SEQ ID NO: 35)
GAATGATTAAACC


 



RPldhA3
CAAAAAATTCCAGCTCAAAGCCAAAGGACTCGTTCACCTGTTGCAGG



(SEQ ID NO: 36)
TACTTCTTGTCGTACTGTTTTGTG


 


acs
FPacs1
TCACGACAGTAACCGCACCTACACTGTCATGACATTGCTCGCCCCTA



(SEQ ID NO: 37)
TGTGTAACAAATA


 



RPacs1
TGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAAT



(SEQ ID NO: 38)
TGTCAACAGCTAGTGGATCTGATGGGTACC


 



FPacs2
TCACGACAGTAACCGCACCTACACTGTCATGACATTGCTCGCCCCTA



(SEQ ID NO: 39)
TGTGTAACAAATA


 



RPacs2
CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATGGTCTGT



(SEQ ID NO: 40)
TTCCTGTGTGAAATTGTTATC CGCTCACAATTCC


 



FPacs3
CGAATTGCGCCATTGTTGCAATGGCGGTTTTTATTGTTTTTCACGAC



(SEQ ID NO: 41)
AGTAACCGCACCT


 



RPacs3
TTGTTGATACATCGCCTCGTACTGCTGAGGGTTTATCAGGCAACGGT



(SEQ ID NO: 42)
CTGCGATGTTGGCAGGAATGGTG


 


PLGA




K.O.




iclr
11HLiclR-
TGAAAATGATTTCCACGATACAGAAAAAAGAGACTGTCATGGTCGCA



inXB:
CCCATTCCGCATCAGAGCAGATTGTACTGAGAG



(SEQ ID NO: 43)



 



12HLiclR-inXB:
TGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGTCAGCGCATT



(SEQ ID NO: 44)
CCACCGTACCTGATTCTGTGGATAACCGTATTAC


 


K.O. +




PaceA




aceB-A
11HLaceB-A_PC
GCGGATAAATCGCTGGAAGCCAATAACGGTCACGATGGCACATGGAT



inXB:
CGCCGCGTCATACACATACGATT



(SEQ ID NO: 45)



 



12HRaceB-A PC-
GGTTGAGTCCACTCTTTCTGTAATTCTTCAATTTGTTGTGTACGGGT



inXB:
TTTCATGGTCTGTTTCCTGTGTG



(SEQ ID NO: 46)



 


ycdWF
ycdWf-EcoRI
gaattc atggatatcatcttttatcac



(SEQ ID NO: 47)



 



ycdWb-KpnI
ggtacc ttagtagccgcgtgcgcggtc



(SEQ ID NO: 48)



 


hprAf
hprAf-EcoRI
gaattc atgcccagcccgcgtcgcgc



(SEQ ID NO: 49)



 



hprAb-KpnI
ggtacc tcagctgaccacgcggcgtg



(SEQ ID NO: 50)









Cell Growth and Metabolite Analysis
Metabolites including glucose, pyruvic acid, acetic acid, formic acid, lactate, and succinate were analyzed by HPLC (Varian ProStar 210, USA) equipped with UV/VIS (Varian ProStar 320, USA) and refractive index (Shodex RI-71, Japan) using a MetaCarb 87H column (300×7.8 mM). Cell growth was measured at 600 nm absorbance using Ultraspec 300 spectrophotometer (Amersham Bioscience, Sweden).
Example 2
Biosynthesis of P(LA-co-GA) Using E. coli Expressing PHA Synthase, Propionyl-CoA Transferase, and Glycerate Dehydrogenase
In the present Example, in order to construct a recombinant E. coli producing P(LA-co-GA) in which a glycolate fraction is increased, genes to be inserted were selected.
It was confirmed through previous study (Korean Patent Laid-Open Publication No. 2010-0111766) that E. coli transfected with enzyme (Pct 540) having V193A mutation and 4 silent mutations of T78C, T669C, A1125G, and T1158C in the propionate CoA-transferase of Clostridium propionicum and enzyme having quadruple mutation PhaC1437 (E130D, S325T, S477G, and Q481K) in the PHA synthase of Pseudomonas sp. 6-19 may produce the P(LA-co-GA) having a high lactate fraction, and in order to synthesize the P(LA-co-GA), two mutated enzymes were selected.
First, in order to prepare a polymer containing glycolate, sodium glycolate was added to the medium at a concentration of 2 g/L as a precursor of glycolyl-CoA. It was reported that glycolate was used as a single carbon source in E. coli, but the concentration of the glycolate in the medium was not decreased during a culture period. When 3HB and glycolate were added to the medium at a concentration of 2 g/L, respectively, glycolate was not used to synthesize P(3HB-co-LA). This result means that a glycolate transport system mediated by glycolate permease (GlcA) did not operate under the present experimental condition.
Therefore, in order to overcome the problem that glycolate was not transported into the cells, the present inventors allowed glycolate to be produced from glyoxylate by glyoxylate reductase/glycerate dehydrogenase (FIG. 1B)
A reaction of converting glycerate into glycolate was performed by enzyme commission (EC) Nos. 1.1.1.79 (NAD(P)H-dependent glyoxylate reductase), 1.1.1.29 (NADH-dependent glyoxylate reductase), or 1.1.1.26 (glycerate dehydrogenase). Productivity of glycolate for synthesizing the PLGA copolymer from glucose was confirmed using NADPH-dependent glyoxylate reductase (EC 1.1.1.79) genes of E. coli MG1655 purchased from ATCC and KCTC and E. coli MG1655 strains amplified from chromosome of P. putida KT2440 and P. multocida and glycerate dehydrogenase (EC 1.1.1.26) genes of P. putida KT2440 and P. multocida. 
As a result, among these genes, only glycerate hydrogenase (SEQ ID No: 51) of E. coli and glycerate hydrogenase (SEQ ID No: 2) of P. multocida may produce the PLGA copolymer, but in the case of using the glycerate hydrogenase of P. multocida, since the GA fraction in the polymer was higher than in the case of using the glycerate hydrogenase of E. coli, the glycerate hydrogenase of P. multocida was used in the following Experiments.










TABLE 3









Polymer




composition
Polymer



(mol %)
concentration











recombinant strain
polymer
LA
GA
(wt %)





XB/pPs619C1437-
P(LA-co-GA)
47.8
52.2
12.3


CpPCT540 +


pTac15kPmu05591


XB/p619C1437-
P(LA-co-GA)
61.4
38.6
12.8


PCT540 + pKM22-ycdW









Recombinant E. coli XL-1 Blue strains expressing Pct540Cp, PhaC1437Ps6-19, and the glycerate dehydrogenase of P. multocida produced P(47.8 mol % LA-co-52.2 mol % GA) at a polymer concentration of 12.3 weight % (Table 3).
The PHA synthase of Pseudomonas sp. 6-19 did not efficiently produce a polymer containing lactate without 3HB, which is a monomer having the highest affinity in synthesis of the polymer, which may be caused by low substrate-specificity to lactyl-CoA and a restricted content of lactyl-CoA in wild-type E. coli strain. It was thought that 3HB-CoA maybe serves as an initiator in synthesizing the polymer containing lactate (Yang et al., Biotechnol. Bioeng., 105; 150, 2010). However, at the time of synthesizing PLGA, even in the case of not adding 3HB, the lactate monomer was efficiently contained in the PLGA at 50 mol % or more, such that it was confirmed that the glycolyl-CoA was a substrate having significant affinity for PhaC1437Ps6-19 and may be used as an initiator such as 3HB. The glycolyl-CoA was produced by the recombinant E. coli expressing glycerate dehydrogenase of P. multocida shown in Table 2.
As a result, it may be appreciated that an enzyme activity of glycerate dehydrogenase of P. multocida plays an important role in synthesizing glycolate from glycerate.
In measuring the enzyme activity, after 10 hours and 70 hours of 1 mM isopropylthiogalactoside (IPTG) induction, the recombinant E. coli was sampled and centrifuged at 10,000×g for 5 minutes to obtain a cell pellet. Then, the obtained cell pellet was washed with a TDM buffer (50 mM Tris hydrochloride, 10 mM MgCl2, and 1 mM dithiothreitol, pH 7.5) two times, and suspended in the same buffer, followed by destructing cells using a titanium probe 40T (diameter: 4 mm, length: 127 mm) of an ultrasonic homogenizer (VCX-600, Sonics and Materials Inc., USA). Then the resultant was centrifuged at 4° C. and 16,000×g for 10 minutes, and the supernatant was used to measure the enzyme activity. A protein concentration of the cell extracts for measuring the enzyme activity was measured by a Bradford method.
In this case, the enzyme activity was an average value obtained by repeating the measuring experiment 3 times, and the recombinant JLX10 strain was cultured at 30° C. or 72 hours using a MR medium to which 20 g/L glucose and 4 g/L succinate were added. The recombinant XL1-Blue strain was cultured in a MR medium to which only 20 g/L glucose was added.
The sample for analyzing the enzyme activity was extracted after 10 hours and 70 hours of 1 mM isopropylthiogalactoside (IPTG) induction. 1 unit of enzyme activity was defined as the amount of enzyme converting a 1 mmole of a substrate into a specific product at 30° C. for 1 minute. A unit of activity was defined as unit/mg protein.
The enzyme activity was analyzed at 30° C. using a temperature control spectrophotometer (SpectraMax M2, Molecular Devices Co., USA). The enzyme activity was repeatedly tested 3 times and analyzed, and an amount of cell extract solution used in analysis was 16.5 μL/mL. Analysis of the reaction was performed by monitoring NAD(P)H at 340 nm, a value used as an extinction coefficient of NAD(P)H at 340 nm was 6.23/mM/cm, and 1 unit of enzyme activity was defined as the amount of enzyme converting a 1 mmole of a substrate into a specific product at 30° C. for 1 minute. A unit of activity was defined as unit/mg protein.
The activity of the glycerate dehydrogenase of P. multocida was analyzed by a method suggested by Rintalls et al, (Rintalla et al., Yeast, 24, 129-136, 2007). A reaction was carried out by adding glyoxylate to the reaction mixture containing 50 mM sodium phosphate buffer (pH 7.0), the cell extracts, and 0.2 mM NAD(P)H so that a concentration of glyoxylate became 25 mM, and the reaction mixture to which glyoxylate was not added was used as a control group.
The wild-type E. coli XL1-Blue had significantly low glycerate dehydrogenase activity of 0.06 U/mg, but the recombinant strain transfected with a plasmid over-expressing glycerate dehydrogenase of P. multocida had glycerate dehydrogenase activity of 1.4 U/mg protein or more, which was 23 times higher than that of the wild-type E. coli strain.
In the case of using NADH as a cofactor, the enzyme activity of glycerate dehydrogenase of P. multocida was measured, but in the case of using NADPH as a cofactor, an enzyme reaction was not carried out, such that it may be appreciated that the glycerate dehydrogenase of P. multocida did not have an enzyme activity for NADPH.







TABLE 4







Activity of glycerate dehydrogenase of P. multocida


Concentration











A unit of acivity of





glycerate
Polymer



dehydrogenase *
composition
Polymer



(U/mg of protein)
(mol %)
concentration












Recombinant strain
10 h
70 h
LA
GA
(weigh %)





XL1-
0.06
0.03
—
—
—


Blue/pBluescript +


pTac15k


XL1-Blue/
1.60
1.40
49
51
13.5


pPs619C1437-


CpPCT540 +


pTac15kPmu0559









In producing the polymer containing lactate in the recombinant strain, since a step of producing lactyl-CoA is a rate-limiting step deteriorating polymer production efficiency, in order to increase the content of the lactate in the strain, a JLXF5 strain in which several metabolic pathways competitively using the lactate precursor were deleted in the chromosome of E. coli XL1-Blue was constructed. In addition, a lacIq gene under a control of a trc promoter was also deleted (See Table 1 and FIGS. 1 to 4).
As shown in Table 5, the E. coli JLXF5 strain expressing Pct540, PhaC1437, and glycerate dehydrogenase of P. multocida produced P(57.2 mol % LA-co-42.8 mol % GA) at a polymer concentration of 20.3 weight % or more without IPTG induction.
When the concentration of the lactate in the strain was increase, the lactate fraction in PLGA was increased. In the case of adding IPTG, the polymer concentration was increased from 20.3 to 26.9 weight %, and a composition of the monomer was barely changed.







TABLE 5







Composition and concentration of PLAG copolymer


produced in recombinant E. coli


Polymer concentration










Polymer
Polymer



composition
con-



(mol %)
centration











Recombinant strain
polymer
LA
GA
(wt %)














XB/pPs619C1437-CpPCT540 +
P(LA-co-
47.8
52.2
12.3


pTac15kPmu05591
GA)


JLXF5/pPs619C1437-
P(LA-co-
54.6
45.4
26.9


CpPCT540 + pTac15kPmu05591
GA)


JLXF5/pPs619C1437-
P(LA-co-
57.2
42.8
20.3


CpPCT540 + pTac15kPmu0559
GA)


JLXF6/pPs619C1437-
P(LA-co-
43.7
56.2
23.9


CpPCT540 + pTac15kPmu0559
GA)


JLXF7/pPs619C1437-
P(LA-co-
40.2
59.8
29.6


CpPCT540 + pTac15kPmu0559
GA)


JLXF8/pPs619C1437-
P(LA-co-
38.4
61.6
32.6


CpPCT540 + pTac15kPmu0559
GA)









In order to express ldhA and acs genes of chromosome under a control of a 1trc promoter and genes of glycerate dehydrogenase of P. multocida under a control of a tac promoter of the plasmid, when a concentration of the culturing solution was 0.5 of OD600, 1 mM IPTG was treated.
Example 3
Production of PLGA Having High Glycolate Content Through Metabolic Engineering of E. coli 
The PLGA was successfully produced from glucose using the recombinant XL1-Blue strains and recombinant JLXF5 strains in Example 2, but a concentration of the produced polymer was low. In order to improve PLGA biosynthesis in the recombinant strains, a glycolate synthesis pathway was enhanced in the JLXF5 strain modulated so as to efficiently produce lactate from glucose. In addition, an increase in the glycolate content may be advantageous for allowing glycolyl-CoA to start synthesis of the polymer. In order to increase synthesis of glycolate, the aceB and iclR genes involved in glyoxylate shunt were deleted, and the aceA gene was amplified.
In order to increase a glyoxylate pathway, a one-step inactivation method (Datsenko and Wanner, 2000) of deleting the iclR (isocitrate lyase regulator) and aceB (malate synthase) gene in the chromosome DNA and replacing the promoter of aceA (isocitrate lyase) gene with the trc promoter was used.
When the iclR gene was deleted, the concentration of PLGA produced in the recombinant strain was increased from 14.2 to 23.9 weight %, and the glycolate monomer fraction in the PLGA polymer was increased from 42.8 mol % to 56.2 mol %. In the case of additionally deleting the aceB gene and amplifying aceA gene, the concentration of PLGA produced in the recombinant strain was 32.6 weight %, which was 2.3 times higher than that of PLGA produced in JLFX5 strain. Further, the glycolate monomer was increased to 61.6 mol % or more.
Example 4
Characteristics of PLGA Synthesized in Recombinant E. coli 
A monomer component of the copolymer synthesized in the present invention was analyzed using a gas chromatography, and the polymer was purified from cells by an organic solvent extraction method (Jacque et al., Biochem. Eng J 39:15-27, 2008). A molecular weight of the polymer was measured using nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC).
The PLGA produced by the E. coli JLXF5 had the same structure as that of chemically synthesized PLGA (See FIG. 1A). Ina 500 MHz 1H NMR spectrum of the PLGA, an oxymethine proton (—OCH—) of LA was shown at 5.2 ppm and a methyl proton (—CH3) of GA was shown at 4.6 to 4.9 ppm (FIG. 5A).
Internal 3-bond coupling of protons was confirmed in 1H-1H COSY spectrum, and the structure of PLGA became clear therefrom. Coupling between the oxymethine proton of LA and a methylene proton adjacent thereto was confirmed as a cross peak at 5.1 ppm/1.6 ppm (FIG. 5B). Coupling between methylene protons of GA was confirmed as a cross peak present at 4.6 to 4.9 ppm. In a 125 MHz 13C NMR spectrum of PLGA, a carbonyl carbon peak of GA*-GA sequence was shown at 169.4 ppm, a carbonyl peak of LA*-LA and LA-LA* sequences was shown at 169.63 ppm, and a carbonyl peak of LA*-GA+GA-LA* sequences was shown at 169.80 ppm (FIG. 5C).
INDUSTRIAL APPLICABILITY
As set forth above, according to the present invention, the poly(lactate-co-glycolate) in which the concentration of the glycolate fraction is high may be prepared at a high concentration without supplying exogenous glyoxylate.
Although the present invention has been described in detail based on particular features thereof, and it is obvious to those skilled in the art that these specific technologies are merely preferable embodiments and thus the scope of the present invention is not limited to the embodiments. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalent thereof.

## Claims

1. A recombinant microorganism having producibility of poly(lactate-co-glycolate), wherein the microorganism is transfected so that a gene coding poly(hydroxyalkanoate) (PHA) synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted.

2. The recombinant microorganism of claim 1, wherein the propionate CoA-transferase is propionate CoA-transferase of C. propionicum or Pct540, which is a mutant enzyme of propionate CoA-transferase of C. propionicum.

3. The recombinant microorganism of claim 1, wherein the poly(hydroxyalkanoate) (PHA) synthase is PHA synthase of Pseudomonas sp. 6-19 or a mutant enzyme of PHA synthase of Pseudomonas sp. 6-19.

4. The recombinant microorganism of claim 3, wherein the gene coding the mutant enzyme of the PHA synthase has a base sequence corresponding to an amino acid sequence including at least one mutation selected from a group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in an amino acid sequence of SEQ ID No: 1.

5. The recombinant microorganism of claim 3, wherein the gene coding the mutant enzyme of the PHA synthase has a base sequence corresponding to an amino acid sequence selected from a group consisting of:
an amino acid sequence (C1335) in which E130D, S325T, L412M, S477G, and Q481M in the amino acid sequence of SEQ ID No: 1 are mutated;
an amino acid sequence (C1310) in which E130D, S477F, and Q481K in the amino acid sequence of SEQ ID No: 1 are mutated; and
an amino acid sequence (C1312) in which E130D, S477F, and Q481R in the amino acid sequence of SEQ ID No: 1 are mutated.

6. The recombinant microorganism of claim 1, wherein the gene coding glycerate dehydrogenase (EC 1.1.1.26) is derived from Pasteurella multocida.

7. The recombinant microorganism of claim 6, wherein the gene coding glycerate dehydrogenase has a base sequence of SEQ ID No: 2.

8. A recombinant microorganism having producibility of poly(lactate-co-glycolate), wherein the microorganism is transfected so that a gene coding PHA synthase, a gene coding propionyl-CoA transferase, and a gene coding glycerate dehydrogenase (EC 1.1.1.26) are inserted, and a gene (iclR) coding isocitrate lyase regulator or aceB (malate synthase) gene is deleted.

9. The recombinant microorganism of claim 8, wherein the propionate CoA-transferase is propionate CoA-transferase of C. propionicum or Pct540, which is a mutant enzyme of propionate CoA-transferase of C. propionicum.

10. The recombinant microorganism of claim 8, wherein the poly(hydroxyalkanoate) (PHA) synthase is PHA synthase of Pseudomonas sp. 6-19 or a mutant enzyme of PHA synthase of Pseudomonas sp. 6-19.

11. The recombinant microorganism of claim 10, wherein the gene coding the mutant enzyme of the PHA synthase has a base sequence corresponding to an amino acid sequence including at least one mutation selected from a group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in an amino acid sequence of SEQ ID No: 1.

12. The recombinant microorganism of claim 10, wherein the gene coding the mutant enzyme of the PHA synthase has a base sequence corresponding to an amino acid sequence selected from a group consisting of:
an amino acid sequence (C1335) in which E130D, S325T, L412M, S477G, and Q481M in the amino acid sequence of SEQ ID No: 1 are mutated;
an amino acid sequence (C1310) in which E130D, S477F, and Q481K in the amino acid sequence of SEQ ID No: 1 are mutated; and
an amino acid sequence (C1312) in which E130D, S477F, and Q481R in the amino acid sequence of SEQ ID No: 1 are mutated.

13. The recombinant microorganism of claim 10, wherein the gene coding glycerate dehydrogenase (EC 1.1.1.26) is derived from Pasteurella multocida or E. coli.

14. The recombinant microorganism of claim 13, wherein the gene coding glycerate dehydrogenase has a base sequence of SEQ ID No: 2.

15. The recombinant microorganism of claim 10, wherein both of the gene (iclR) coding isocitrate lyase regulator and a gene (aceB) coding isocitrate lyase are deleted.

16. The recombinant microorganism of claim 10, wherein the gene (aceA) coding isocitrate lyase is additionally amplified.

17. A method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing the recombinant microorganism of claim 1 to produce poly(lactate-co-glycolate); and obtaining the produced PLGA.

18. A method of preparing poly(lactate-co-glycolate) (PLGA) including: culturing the recombinant microorganism of claim 10 to produce poly(lactate-co-glycolate); and obtaining the produced PLGA.