WO2011074842A2

WO2011074842A2 - Recombinant microorganism for producing polylactic acid or a polylactic acid copolymer from glycerol and method for producing polylactic acid or a polylactic acid copolymer from glycerol using the microorganism

Info

Publication number: WO2011074842A2
Application number: PCT/KR2010/008901
Authority: WO
Inventors: 김태완; 강혜옥; 양택호
Original assignee: (주)Lg화학
Priority date: 2009-12-14
Filing date: 2010-12-13
Publication date: 2011-06-23
Also published as: WO2011074842A3

Abstract

The present invention relates to a recombinant microorganism for producing a lactate copolymer or a copolymer using glycerol and to a method for producing a lactate copolymer or a copolymer using glycerol as one of substrates and using the microorganism. According to the present invention, genes of the enzyme which converts lactate into lactile-CoA, and genes of polyhydroxyalkanoate (PHA) synthase which uses lactile-CoA as a substrate are introduced to the microorganism which can use glycerol as a substrate, and the microorganism is then cultured using the glycerol as a substrate to efficiently produce a lactate copolymer and a copolymer.

Description

Recombinant microorganisms capable of producing polylactic acid or polylactic acid copolymers from glycerol and methods of preparing polylactic acid or lactic acid copolymers from glycerol using such microorganisms

The present invention relates to recombinant microorganisms capable of producing polylactic acid or polylactic acid copolymers from glycerol and to methods of producing polylactic acid or lactic acid copolymers from glycerol using such microorganisms.

Polylactate (PLA) is a representative biodegradable polymer derived from lactate and is a polymer having high applicability as a general purpose polymer or a medical polymer. Currently, PLA is produced by polymerizing lactate produced by microbial fermentation, but only low molecular weight (1000-5000 Daltons) PLA is produced by the direct polymerization of lactate. In order to synthesize more than 100,000 Daltons of PLA, there is a method of polymerizing from PLA of low molecular weight obtained by direct polymerization of lactate to PLA of higher molecular weight using a chain coupling agent, but using an organic solvent or a chain couple. The addition of ring agents complicates the process and also has the disadvantage that it is not easy to remove them. Currently commercially available high molecular weight PLA production process is used to convert the lactate to lactide (lactide), and then synthesize the PLA through the ring-opening condensation reaction of the lactide ring.

PLA homopolymers can be easily obtained when PLA is synthesized using lactate through chemical synthesis, but the synthesis of PLA copolymers having various monomer compositions is difficult and commercially useful.

On the other hand, polyhydroxyalkanoaste (PHA) is a polyester (microorganisms) that accumulate inside as energy or carbon storage material when there is an excessive carbon source and lacks other nutrients such as phosphorus, nitrogen, magnesium and oxygen. polyester). PHA is regarded as a material to replace conventional synthetic plastics because it has properties similar to synthetic polymers derived from petroleum and shows complete biodegradability.

In order to produce PHA in microorganisms, enzymes for converting metabolites of microorganisms to PHA monomers and PHA synthase synthesizing PHA polymers using PHA monomers are essential. The same system is required when synthesizing PLA and LA copolymers using microorganisms and in addition to enzymes that can provide hydroxyacyl-CoA, the substrate of the original PHA synthase, in addition to lactyl-CoA (lactyl). There is a need for an enzyme capable of providing -CoA).

Furthermore, it is very important to use a low-cost substrate for the economic production of biodegradable polymers, and in particular, a technique for preparing a lactate polymer or a lactate copolymer using a low-cost substrate glycerol is required.

Therefore, the problem to be solved by the present invention is to provide a microorganism capable of producing a lactate polymer or copolymer using glycerol as a substrate and a method for producing a lactate polymer or copolymer using these microorganisms.

In order to achieve the above object, the present invention includes the gene of the enzyme for converting lactate to lactyl-CoA and the gene of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate, Cells or plants having the ability to produce lactate polymers or hydroxyalkanoate-lactate copolymers capable of using glycerol as substrates include lactate and glycerol; Or cultivated or grown in an environment containing lactate, glycerol and hydroxyalkanoate;

Recovering the lactate polymer or hydroxyalkanoate-lactate copolymer from the cell or plant

Provided are methods for preparing lactate polymers or hydroxyalkanoate-lactate copolymers from glycerol.

The inventors of the present invention have found that polyhydroxyalcohol of Pseudomonas sp. 6-19 using propionyl-CoA transferase derived from Clostridium propionicum to provide lactyl-CoA and lactyl-CoA produced thereby as a substrate. Variants of canoate synthase have been used to successfully synthesize lactate polymers and copolymers (Korean Patent Application No. 10-2006-0116234).

Furthermore, the present inventors was to prepare a lactate polymer and a copolymer using a matrix of glycerol, a low cost for the economical production of the biodegradable polymer, and thus, the present inventors have found that the E. coli using the substrate, glycerol-cost Clostridium propionicum It was transformed with a plasmid expressing propionyl-CoA transferase derived from polyhydroxyalkanoate synthase of Pseudomonas sp. 6-19 and transformed with E. coli. It was confirmed from the present invention that lactate polymers and copolymers can be efficiently produced, thereby completing the present invention.

The polylactate or lactate copolymer (hydroxyalkanoate-lactate copolymer: a cell or plant having a poly (hydroxyalkanoate-co-lactate) production ability (a) converts lactate to lactyl-CoA A cell or plant that does not contain any one or more of the gene of the enzyme and (b) the gene of PHA synthase using r-lactyl-CoA as a substrate, the gene of any one or more of (a) and (b) Can be obtained by transforming a cell or plant without (a) and (b) genes into (a) and (b) genes, or (a) without a gene (b) A cell or plant having a gene may be obtained by transformation with the gene (a), but is not limited thereto, for example, in a cell having any one of the above (a) and (b) genes. Amplifying genes and children It is also within the scope of the present invention to transform into other genes.

In the present invention, the hydroxyalkanoate in the hydroxyalkanoate-lactate copolymer is 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxy 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 -hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid (3-hydroxyhexadecanoic acid), 4-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxy Octanoic acid (4-hydroxyoctanoic acid), 4-hydroxydecanoic acid, 5-hydroxyvaleric acid, 5-hydroxyhexanoic acid, 6- 6-hydroxydodecanoic acid, 3-hydroxy-4-pentenoic acid, 3-hydroxy-4-trans-hexenoic acid (3-hydroxy-4-trans- hexenoic acid), 3-hydroxy-4-cis-hexenoic acid, 3-hydroxy-5-hexenoic acid, 3- 3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy 3-hydroxy-7-octenoic acid, 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid 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-methylvaleric acid acid), 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methylhexanoic acid, 3-hydroxy- 3-hydroxy-6-methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid hydroxy-5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3-hydroxy-6-methyl 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid -methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy 3-hydroxy-7-methyl-6-octennoic acid, malic acid, 3-hydroxysuccinic acid-methyl ester, 3 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelinic acid methyl ester hydroxyazelaic acid-methyl ester, 3-hydroxysebacic acid-methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxy Sebacic acid-ethyl ester (3-hydroxysebac ic acid-ethyl ester, 3-hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzil ester, 3-hydroxy 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyrate -3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxy Phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid cyanophenoxy-3-hydroxyvaleric acid), para-cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid (para-nitrophenoxy-3 -h ydroxyhexanoic acid), 3-hydroxy-5-phenylvaleric acid, 3-hydroxy-5-cyclohexylbutyric acid, 3,12- Dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy- 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-6,7-epoxydodecanoic acid, 3-hydroxy 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid -cyano-3-hydroxyheptanoic acid), 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid acid), 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy acid -8-clock 3-hydroxy-8-chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid 8-bromooctanoic acid), 3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 6- 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid (3 -hydroxy-2-methylvaleric acid), and at least one hydroxyalkano selected from the group consisting of 3-hydroxy-2,6-dimethyl-5-heptenic acid. It may be an eight but is not limited thereto.

In the present invention, as the gene of the enzyme for converting lactate to lactyl-CoA, propionyl-CoA transferase gene ( pct ) may be used, and more specifically, such lactate may be lactyl-CoA. The gene of the enzyme converting to may be a propionyl-CoA transferase gene derived from Clostridium propionicum . In one embodiment of the invention, the gene of the enzyme for converting lactate to lactyl-CoA is the nucleotide sequence of SEQ ID NO: 1 (CpPCT); Nucleotide sequences of which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1 (CpPCT522); A nucleotide sequence of A1200G mutated from the nucleotide sequence of SEQ ID NO: 1 (CpPCT512); A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and Gly335Asp is mutated in the amino acid sequence of SEQ ID NO: 2 (CpPCT531); A base sequence (CpPCT533) in which T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Asp65Gly is mutated in the amino acid sequence of SEQ ID NO: 2; A base sequence (CpPCT535) in which T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Asp65Asn is mutated in the amino acid sequence of SEQ ID NO: 2; A base sequence (CpPCT537) in which T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Thr199Ile is mutated in the amino acid sequence of SEQ ID NO: 2; A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1, and Ala243Thr is mutated in the nucleotide sequence of SEQ ID NO: 2 (CpPCT532); A nucleotide sequence in which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and Asp257Asn is mutated in the nucleotide sequence of SEQ ID NO: 2 (CpPCT534); T78C, T669C, A1125G and T1158C are modified in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala in the amino acid sequence of SEQ ID NO: 2 may be a gene having a nucleotide sequence (CpPCT540).

In particular, the CpPct540 gene is a gene of the Clostridium propionicum- derived propionyl-CoA transferase variant which is more preferred for producing lactate polymers or copolymers using glycerol.

The cell or plant according to the invention also comprises a gene of PHA synthase (Polyhydroxyalkanoate synthase) using the lactyl-CoA as a substrate. Such PHA synthase genes include the amino acid sequence of SEQ ID NO: 4 which is a PHA synthase derived from Pseudomonas genus 6-19; Or a gene having a base sequence corresponding to an amino acid sequence comprising one or more mutations selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in the amino acid sequence of SEQ ID NO: 4 And the like can be used.

In particular, the phaC1 _Ps6-19 337 gene is a gene of the PHA synthase variant derived from Pseudomonas 6-19 which is more preferable for producing lactate polymers or copolymers using glycerol.

In addition, the cell or plant according to the present invention may further include a gene of an enzyme that generates hydroxyacyl-CoA from glycerol. Recombinant cells or plants that additionally contain the gene of the enzyme that produces hydroxyacyl-CoA from glycerol can produce hydroxyacyl-CoA by itself, so do not include hydroxyalkanoate in the medium. If not, it is possible to produce hydroxyalkanoate-lactate copolymers in high yields. In one embodiment of the present invention, the enzyme for producing hydroxyacyl-CoA from the glycerol may be ketothiolase and acetoacetyl-CoA reductase, but is not limited thereto. The ketothiolase and acetoacetyl-CoA reductase are preferably derived from Ralstonia eutropha .

In one embodiment of the invention, the cells having the ability to produce lactate polymers or hydroxyalkanoate-lactate copolymers can be bacteria, in particular E. coli.

The invention also relates to lactate polymers or copolymers comprising genes of enzymes that convert lactate to lactyl-CoA and genes of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate. Provided are cells or plants which can use glycerol transformed with a recombinant vector for preparation as a substrate. The cell or plant may further comprise a gene of an enzyme that produces 3-hydroxybutyl-CoA from glycerol.

The term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. In the present invention, a plasmid vector, a bacteriophage vector, a cosmid vector, a YAC (Yeast Artificial Chromosome) vector, and the like may be used. Preference is given to using plasmid vectors for the purposes of the present invention. Typical plasmid vectors that can be used for such purposes include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Preferred host cells for the present invention may be prokaryotic or eukaryotic cells. Preferred host cells are prokaryotic cells. Suitable prokaryotic cells can be used as well as microorganisms having any of the three genes described above, as well as microorganisms which do not have all of these genes such as E. coli. Preferred E. coli include E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the microorganism having the gene of E. coli and PHA synthase described above, Agrobacterium A4 and Agrobacterium sp, Bacillus subtilis (Bacillus subtilis) and Bashile (bacilli), S. typhimurium (Salmonella such as Another enterobacteria such as typhimurium ) or Serratia marcescens may be used as the host cell. Known eukaryotic host cells such as yeast and fungi, insect cells such as Spodoptera fruitgiper (SF9), animal cells such as CHO and mouse cells, tissue cultured human cells and plant cells can also be used. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself.

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

The expression “expression control sequence” refers to a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from cytomegalovirus, and the like are preferably used.

To express the DNA sequences of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter region of phage lambda, fd Regulatory regions of the code protein, promoters for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoters of the yeast alpha-crossing system and prokaryotic or eukaryotic cells or viruses thereof And other sequences of constitution and induction known to modulate the expression of the genes, and various combinations thereof.

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

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. Within the scope of these variables, one skilled in the art can select various vector / expression control sequence / host combinations suitable for the present invention.

In addition, prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al ., Supra. Optionally, electroporation (Neumann et al., EMBO J., 1: 841 (1982)) can also be used to transform these cells.

On the other hand, transformation of plants can be achieved by conventional methods using Agrobacterium, viral vectors and the like. For example, after transforming the microorganism of the genus Agrobacterium with a recombinant vector containing the gene according to the present invention, the transformed plant can be obtained by infecting the transformed Agrobacterium microorganisms in the tissues of the target plant. For example, a transgenic plant suitable for the present invention can be obtained by the same or similar method as in the prior patent (WO 94/11519; US 6,103,956) for producing PHA using the transformed plant. More specifically, (a) pre-culture the explant of the plant (preplant), and then transfected by co-culture with the transformed Agrobacterium; (b) culturing the transfected explants in a callus induction medium to obtain callus; And (c) cutting the obtained callus, and culturing it in shoot induction medium to form shoots, thereby producing a transfected plant.

In the present invention, the term "explant" refers to a slice of tissue cut out of a plant, and includes cotyledon or hypocotyl. The explants of the plant used in the method of the present invention may be cotyledons or hypocotyls, it is more preferable to use the cotyledons obtained by germinating in MS medium after disinfecting and washing the seeds of the plants.

Plants to be transformed usable in the present invention include tobacco, tomato, pepper, soybean, rice, corn, and the like, but is not limited thereto. In addition, even if the plant used for transformation is a sexually propagating plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture or the like.

The present invention provides cells or plants capable of efficiently producing lactate polymers or copolymers from glycerol, which is a low-cost substrate, and the production of lactate polymers or copolymers comprising cultivating or culturing such cells or plants. Provide a method.

Figure 1 shows the pPs619C1337-CPPCT540 vector.

2 depicts the pMCS104ReAB plasmid.

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

Example 1 Preparation of Recombinant E. Coli

Recombinant Escherichia coli XL1-Blue transformed with pPs619C1337CPPCT540 plasmid and recombinant plasmid Escherichia coli XL1-Blue transformed with two plasmids, pPs619C1337CPPCT540 and pMCS104ReAB, were prepared, respectively. The pPs619C1337CPPCT540 plasmid and pMCS104ReAB were designed to express two major enzymes. These enzymes are essential enzymes for the biosynthesis of the biodegradable polymers polylactate and poly (3-hydroxybutyrate-co-lactate) in Escherichia coli, Pseudomonas sp. PhaC1 _Ps6-19 337, a polymer polymerase derived from 6-19 (KCTC 11027BP), propionyl-CoA transferase derived from Clostridium propionicum , an enzyme that transfers CoA from acetyl-CoA to lactate and converts it into lactic-CoA (propionyl-CoA transferase, CPPCT), and ketothiolase (phaA _RE ) and acetoacetyl-CoA reductase (phaB _RE ) from Ralstonia eutropha , enzymes that synthesize 3-hydroxybutyl-CoA from glycerol. The pPs619C1337CPPCT540 plasmid contains phaC1 _Ps6-19 337 and CPPCT540 genes (FIG. 1), and the pMCS104ReAB plasmid contains phaA _RE and phaB _RE genes (FIG. 2).

In addition, for the phaC1 _Ps6-19 337 gene and the CPPCT540 gene, SEQ ID NO: 3 Pseudomonas sp. PHA synthase derived from phaC1 _Ps6-19 gene and propionyl-CoA transferase (CPPCT) gene derived from Clostridium propionicum of SEQ ID NO: 1 genes mutated to favor lactate polymer and copolymer, respectively . All of the genes contained on each plasmid were constructed to be constitutively expressed in recombinant E. coli.

Example 1-1 Pseudomonas sp. Substrate Specificity Variation of 6-19-derived PHA Synthetase

Among various types of PHA synthase, Type II PHA synthase is known as medium-chain-length PHA synthase that polymerizes relatively long carbon substrates, and this MCL-PHA synthase produces lactate copolymers. It is expected to be very useful. Pseudomonas sp. Highly homologous to phaC1 _{Ps6-19 synthase} obtained in the present invention. 61-3-derived phaC1 synthase is a Type II synthase, but has been reported to have a relatively broad range of substrate specificities (Matsusaki et al ., J. Bacteriol ., 180: 6459, 1998), and SCL-PHA (short-chain). The results of studies on mutants suitable for the production of -length PHA) have been reported (Takase et al ., Biomacromolecules , 5: 480, 2004). Based on this, the inventors have made phaC1 _Ps6-19 synthetase variants using SDM method of amino acids affecting SCL activity (Domestic Patent Application No. 10-2006-0116234).

More specifically, it was prepared as follows. Pseudomonas sp. To isolate PHA synthase (phaC1 _Ps6-19 ) gene from 6-19 (KCTC 11027BP), Pseudomonas sp. To extract the entire DNA of 6-19, to prepare a primer having the base sequence of SEQ ID NO: 5 and 6 based on the phaC1 _Ps6-19 base sequence (Song Aejin, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004) , PCR was performed to obtain phaC1 _Ps6-19 gene.

SEQ ID NO: 5'- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3 '

SEQ ID NO: 5'- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3 '

Agarose gel electrophoresis of the PCR reactions confirmed a 1.7kbp gene fragment corresponding to the phaC1 _Ps6-19 gene. For the expression of phaC1 _{Ps6-19 synthase,} an operon-type constitutive expression system in which a monomer feed enzyme and a synthetase are expressed is introduced.

DNA fragments containing PHB-producing operons from Ralstonia eutropha H16 from pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44: 1337-1347) were digested with BamHI / EcoRI and BamHI from pBluescript II (Stratagene) PReCAB recombinant vector was prepared by inserting in / EcoRI recognition site.

The pReCAB vector is known to express PHA synthase (phaC _RE ) and monomer feeder enzymes (phaA _RE & phaB _RE ) at all times by the PHB operon promoter and to work well in Escherichia coli (Lee et al ., Biotech. Bioeng . , 1994, 44: 1337-1347. The pReCAB vector was digested with BstBI / SbfI to remove R.eutropha H16 PHA synthase (phaC _RE ), and then the pPs619C1-ReAB recombinant vector was prepared by inserting the phaC1 _Ps6-19 gene obtained above into the BstBI / SbfI recognition site. .

In order to make a phaC1 _Ps6-19 synthase gene fragment containing only one BstBI / SbfI recognition site at each end, the BstBI position, which is inherent in the BstBI / SbfI recognition region, was first removed without using amino acid conversion by SDM (site directed mutagenesis) method. In order to add a site, overlapping PCR was performed using primers having the nucleotide sequences of SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NO: 11, and SEQ ID NO: 12.

SEQ ID NO: 5'- atg ccc gga gcc ggt tcg aa-3 '

SEQ ID NO: 5'- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC-3 '

SEQ ID NO: 5'- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3 '

SEQ ID NO: 10'-5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3 '

SEQ ID NO: 5'- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3 '

* SEQ ID NO 12: 5'- aac ggg agg gaa cct gca gg-3 '

The nucleotide sequence of the phaC1 _Ps6-19 gene of the prepared pPs619C1-ReAB recombinant vector was confirmed by sequencing, and is represented by SEQ ID NO: 3, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 4.

PPs619C1-ReAB recombinant vector was transformed into E. coli XL-1Blue (Stratagene) to confirm PHB synthesis of the phaC1 _{Ps6-19 synthase} , and this was detected by PHB detection medium (LB agar, glucose 20g / L, Nile red). At 0.5 μg / ml), no PHB production was observed.

Three amino acid positions affecting SCL (short chain length) activity were found through amino acid sequence analysis, and the phaC1 _Ps6-19 synthetase variants shown in Table 1 were obtained using the SDM method using the primers of SEQ ID NOs: 13-18. Made them.

Table 1

Recombinant vector	Nucleic Acid Substitution	Amino acid substitutions	primer
pPs619C1200-ReAB	AGC → ACC	S325T	SEQ ID NO: 13/14
pPs619C1200-ReAB	CAG → ATG	Q481M	SEQ ID NO: 15/16
pPs619C1300-ReAB	GAA → GAT	E130D	SEQ ID NO: 17/18
	AGC → ACC	S325T	SEQ ID NO: 13/14
	CAG → ATG	Q481M	SEQ ID NO: 15/16

S325T

SEQ ID NO: 5- CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- 3

SEQ ID NO: 14 5- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3

Q481M

SEQ ID NO: 15 5-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- 3

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

E130D

SEQ ID NO: 17 5- atc aac ctc atg acc gat gcg atg gcg ccg acc-3

SEQ ID NO: 18 5- ggt cgg cgc cat cgc atc ggt cat gag gtt gat- 3

These recombinant vectors were transformed into E. coli XL1-Blue, and grown in PHB detection medium (LB agar, glucose 20 g / L, Nile red 0.5 μg / ml), resulting in E. coli transformed with pPs619C1200-ReAB. PHB production was confirmed in both XL1-Blue and E. coli XL1-Blue transformed with pPs619C1300-ReAB. That is, 3HB-CoA is generated from glucose by the monomer supply enzymes phaA _RE and phaB _RE , and phaC1 _Ps6-19 synthase SCL variants (phaC1 _Ps6-19 200 & phaC1 _Ps6-19 300) are used as the substrates. Is synthesized.

Propionyl-CoA derived from Clostridium propionicum to construct a system of constant expression of the operon form in which CP-PCT is expressed together to provide lactyl-CoA, which is a monomer required for the synthesis of PLA and PLA copolymers. Transferase (CP-PCT) was used. cp-pct used a fragment obtained by PCR chromosomal DNA of Clostridium propionicum using primers of SEQ ID NO: 19 and SEQ ID NO: 20, at this time, for ease of cloning the Nde I site existing in the wild type CP-PCT. It was removed using the SDM method.

SEQ ID NO: 19: 5-ggaattcATGAGAAAGGTTCCCATTATTACCGCAGATGA

SEQ ID NO: 20: 5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg

In addition, overlapping PCR was performed using primers having the nucleotide sequences of SEQ ID NOs: 21 and 22 to add Sbf I / Nde I recognition sites.

SEQ ID NO: 21 5-agg cct gca ggc gga taa caa ttt cac aca gg- 3

SEQ ID NO: 22 5-gcc cat atg tct aga tta gga ctt cat ttc c- 3

phaC1 _Ps6-19 synthase remove the SCL mutant of phaC1 _Ps6-19 300 a pPs619C1300-ReAB by cutting the vector with Sbf I / Nde I Ralstonia eutrophus H16 monomer feed enzymes (phaA _{_RE} & phaB _RE) of origin containing the following, the The pPs619C1300-CPPCT recombinant vector was prepared by inserting the PCR cloned CP-PCT gene into the Sbf I / Nde I recognition site.

In a similar manner to the above, various PHA synthase variants were prepared using the following primers. The prepared variants are shown in the following Tables 2 to 5.

E130D

SEQ ID NO: 17'-atc aac ctc atg acc gat gcg atg gcg ccg acc-3 '

SEQ ID NO: 18'-ggt cgg cgc cat cgc atc ggt cat gag gtt gat-3 '

S325T

SEQ ID NO: 5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC-3 '

SEQ ID NO: 14 '5'- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG-3'

S477R

SEQ ID NO: 23'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3 '

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

S477H

SEQ ID NO: 25'-gaa ttc gtg ctg tcg agc cat ggg cat atc-3 '

SEQ ID NO: 26'-gat atg ccc atg gct cga cag cac gaa ttc-3 '

S477F

SEQ ID NO: 27'-gaa ttc gtg ctg tcg agc ttt ggg cat atc-3 '

SEQ ID NO: 28: 5'- gat atg ccc aaa gct cga cag cac gaa ttc-3 '

S477Y

SEQ ID NO: 29 '5'-gaa ttc gtg ctg tcg agc tat ggg cat atc'

SEQ ID NO: 30'-gat atg ccc ata gct cga cag cac gaa ttc-3 '

S477G

SEQ ID NO: 31 5'-gaa ttc gtg ctg tcg agc ggc ggg cat atc-3 '

SEQ ID NO: 32 5'-gat atg ccc gcc gct cga cag cac gaa ttc-3 '

Q481K

SEQ ID NO: 33 5'-ggg cat atc aaa agc atc ctg aac ccg c-3 '

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

Q481M

SEQ ID NO: 35'-ggg cat atc atg agc atc ctg aac ccg c-3 '

SEQ ID NO: 36'-gcg ggt tca gga tgc tca tga tat gcc c-3 '

Q481R

SEQ ID NO: 37: 5'-ggg cat atc cgc agc atc ctg aac ccg c-3 '

SEQ ID NO: 38'-gcg ggt tca gga tgc tgc gga tat gcc c-3 '

TABLE 2

Recombinant synthetase	Nucleic Acid Substitution	Amino acid substitutions	primer
pPs619C1200	AGC → ACC	S325T	SEQ ID NOs: 13, 14
pPs619C1200	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1202	GAA → GAT	E130D	SEQ ID NOs: 17, 18
pPs619C1202	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1203	AGC → ACC	S325T	SEQ ID NOs: 13, 14
pPs619C1203	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1204	GAA → GAT	E130D	SEQ ID NOs: 17, 18
pPs619C1204	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1205	GAA → GAT	E130D	SEQ ID NOs: 17, 18
pPs619C1205	CAG → CGC	Q481R	SEQ ID NOs: 37, 38

TABLE 3

Recombinant synthetase	Nucleic Acid Substitution	Amino acid substitutions	primer
pPs619C1300	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1301	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1304	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CGC	S477R	SEQ ID NOs: 23, 24
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1305	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CGC	S477R	SEQ ID NOs: 23, 24
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1306	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CGC	S477R	SEQ ID NOs: 23, 24
	CAG → CGC	Q481R	SEQ ID NOs: 37, 38
pPs619C1307	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CAT	S477H	SEQ ID NOs: 25, 26
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1308	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CAT	S477H	SEQ ID NOs: 25, 26
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1309	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → CAT	S477H	SEQ ID NOs: 25, 26
	CAG → CGC	Q481R	SEQ ID NOs: 37, 38
pPs619C1310	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TTT	S477F	SEQ ID NOs: 27, 28
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34

Table 4

Recombinant synthetase	Nucleic Acid Substitution	Amino acid substitutions	primer
pPs619C1311	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TTT	S477F	SEQ ID NOs: 27, 28
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1312	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TTT	S477F	SEQ ID NOs: 27, 28
	CAG → CGC	Q481R	SEQ ID NOs: 37, 38
pPs619C1313	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TAT	S477Y	SEQ ID NOs: 29, 30
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1314	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TAT	S477Y	SEQ ID NOs: 29, 30
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1315	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → TAT	S477Y	SEQ ID NOs: 29, 30
	CAG → CGC	Q481R	SEQ ID NOs: 37, 38

Table 5

Recombinant synthetase	Nucleic Acid Substitution	Amino acid substitutions	primer
pPs619C1400	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → CGC	S477R	SEQ ID NOs: 23, 24
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1401	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → CGC	S477R	SEQ ID NOs: 23, 24
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1334	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → TTT	S477F	SEQ ID NOs: 27, 28
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1336	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → GGC	S477G	SEQ ID NOs: 31, 32
	CAG → ATG	Q481M	SEQ ID NOs: 35, 36
pPs619C1337	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → GGC	S477G	SEQ ID NOs: 31, 32
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34
pPs619C1339	GAA → GAT	E130D	SEQ ID NOs: 17, 18
	AGC → ACC	S325T	SEQ ID NOs: 13, 14
	AGC → TTT	S477F	SEQ ID NOs: 27, 28
	CAG → AAA	Q481K	SEQ ID NOs: 33, 34

Example 1-2 Preparation and Screening of Propionyl-CoA Transferase Mutant Library from Clostridium propionicum

CP-PCT is known to be highly toxic due to severe metabolic disorders when expressed in E. coli. In general, the expression of CP-PCT by IPTG using the tac promoter or T7 promoter, which is widely used for expression of recombinant proteins, is recombined simultaneously with the addition of inducers. E. coli all died. This has led to the success of the synthesis of lactate polymers and lactate copolymers using a constitutive expression system that is weakly expressed but continuously expressed as the microorganism grows. pPs619C1300-CPPCT (Korean Patent Application No. 10-2006-0116234) was used as a template to introduce random mutations into cp-pct , Mn ²⁺ was added using primers SEQ ID NOs: 39 and 40, and the concentration of dNTPs Error-prone PCR was performed in the presence of a difference.

SEQ ID NO: 39 '5'-cgc cgg cag gcc tgc agg-3'

SEQ ID NO: 40'-5'-ggc agg tca gcc cat atg tc-3 '

Thereafter, PCR was performed under normal conditions using the primers SEQ ID NOs: 39 and 40 to amplify the PCR fragment containing the random mutation. phaC1 _Ps6-19 synthase SCL mutant of phaC1 by cutting a pPs619C1300-CPPCT vector containing _Ps6-19 300 with Sbf I / Nde I to remove the wild-type cp-pct, the amplified mutant PCR fragment Sbf I / Nde I A ligation mixture was inserted into the recognition site and introduced into E. coli JM109 to produce a CP-PCT library of ~ 10 ^{^ 5} scale. The prepared CP-PCT library was grown in polymer detection medium (LB agar, glucose 20g / L, 3HB 1g / L, Nile red 0.5μg / ml) for 3 days and then screened to determine whether the polymer was produced. More than 80 candidates were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ° C) for 4 days under conditions in which the polymers were produced, and FACS (Florescence Activated Cell Sorting) analysis to analyze the final two individuals. Selected. Gene sequencing was performed to find the mutation position of the prepared CP-PCT variant. The results are shown in Table 6 below.

Table 6

Recombinant vector	Nucleic Acid Substitution
CP-PCT Variant 512	A1200G
CP-PCT Variant 522	T78C, T669C, A1125G, T1158C

Based on the final selected mutants (512, 522) again random mutagenesis by the method of the error-prone PCR was performed to obtain the PCT variants 531-540 as follows.

TABLE 7

	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
CpPct540	Val193Ala	T78C, T669C, A1125G, T1158C

Thereafter, PCR was performed under normal conditions using the primers SEQ ID NOs: 39 and 40 to amplify the PCR fragment containing the CpPct540 mutation. The pPs619C1300-CPPCT vector Sbf I / Nde I was cut to remove the portion CPPCT, the amplified PCR fragments CpPct540 Sbf I / Nde I made the ligation mixture was inserted into the recognition site to prepare a pPs619C1300-CPPCT540 vector.

Example 1-3 Construction of pPs619C1337-CPPCT540 Vector

As summarized in Table 5, the phaC1 _Ps6-19 synthase variant using _{(phaC1 Ps6-19 300) E130D, S325T} , S477G , and Pseudomonas in 6-19-derived PHA having the amino acid sequence of the mutant Q481K synthase variant (phaC1 _{Ps6 -19} 337) was prepared using the SDM method using the primers SEQ ID NOs: 31 and 32, and SEQ ID NOs: 33 and 34, and the pPs619C1337-CPPCT540 vector was constructed using the gene (FIG. 1).

SEQ ID NO: 31 5'-gaa ttc gtg ctg tcg agc ggc ggg cat atc-3 '

SEQ ID NO: 32 5'-gat atg ccc gcc gct cga cag cac gaa ttc-3 '

SEQ ID NO: 33 5'-ggg cat atc aaa agc atc ctg aac ccg c-3 '

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

The recombinant vector (pPs619C1337-CPPCT540) thus obtained was transformed into E. coli JM109, and then transformed into 3HB-containing polymer detecting medium (LB agar, glucose 20g / L, 3HB 2g / L, Nile red 0.5μg / ml). As a result of the growth, the formation of the polymer was confirmed.

Example 1-4 Preparation of pMCS104ReAB

A plasmid pMCS104ReAB was prepared to provide β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) derived from R. eutropha (Pak Si Jae, PhD thesis, Department of Chemical and Biomolecular Engineering). , KAIST, 2003). p10499A (Park et al ., FEMS Microbiol. Lett ., 214: 217, 2002), obtained by cleaving phaAB gene obtained by cleaving pSYL105 (Lee et al . Biotechnol. Bioeng. 44: 1337, 1994) with PstI. By inserting, p10499PhaAB was produced. The p10499PhaAB plasmid was digested with SspI to obtain a gene fragment containing 104 promoter and phaAB gene, and then inserted into pBBR1MCS plasmid digested with EcoRV to prepare pMCS104ReAB plasmid (FIG. 2).

Example 2 Preparation of Polylactate (PLA) from Recombinant Escherichia Coli Using Glycerol as a Carbon Source

One recombinant Escherichia coli XL1-Blue colony transformed with pPs619C1337-CPPCT540 vector as shown on <Example 1-3> on an LB agar plate containing 100 mg / L ampicillin as an antibiotic was treated with 100 mg / L ampicillin. After inoculating 3 mL of LB medium containing it was incubated for 24 hours while stirring at a speed of 200 rpm at 30 ℃. 1 mL of the culture was inoculated into 100 mL of MR medium containing 100 mg / L of ampicillin and 20 g / L or 50 g / L of glycerol, which was incubated at 30 ° C. with a stirring speed of 200 rpm. Started. The culture was carried out for 4 days. In case of MR medium, the initial pH was adjusted to 7 using 10 N NaOH. The medium used for this culture is shown in Table 8 below.

After completion of the culture, cells were recovered from the culture by centrifugation. The recovered cells were washed three times with distilled water, and then dried in a drier at 100 ° C. for 24 hours. Some of the dried cells were collected and subjected to gas chromatography (GC) analysis to synthesize P (3HB-co-LA). ) Content was measured. Standards used in the analysis were P (3HB-co-3HV) copolymer (of which 3HV content was about 12% by weight) and polylactate homopolymer. GC analysis results are shown in Table 9 below. As a result, it was confirmed that PLA homopolymer can be biosynthesized from glycerol. PLA homopolymer can be prepared from glycerol through the recombinant E. coli according to the present invention.

Table 8

ingredient	MR badge (/ L)
KH ₂ PO ₄	6.67 g
(NH ₄ ) ₂ HPO ₄	4 g
Citrate	0.8 g
MgSO ₄ H ₂ O	0.8 g
Sodium lactate	0 g or 2 g
Thiamine	10 mg
Glycerol	20 g or 50 g
Tracer ^*	5 mL

^* Tracer (/ L): FeSO ₄ H ₂ O, 10 g; ZnSO ₄ H ₂ O, 2.25 g; CuSO ₄ H ₂ O, 1 g; MnSO ₄ H ₂ O, 0.5 g; CaCl ₂ H ₂ O, 2 g; Na ₂ B ₄ O ₇ .H ₂ O, 0.23 g; (NH ₄ ) ₆ Mo ₇ O ₂₄ , 0.1 g; 35% HCl, 10 mL.

Table 9

badge	Early temperament	Biosynthetic Polymer Type	Polymer content (weight ratio)	LA content in the polymer (molar ratio)
MR	G2 ^*	PLA	6.48	100
MR	G2, NaL ⁺	PLA	10.00	100
MR	G5 ^**	PLA	1.85	100
MR	G5, NaL	PLA	5.75	100

* G2: Glycerol 20 g / L

** G5: Glycerol 50 g / L

+ NaL: Sodium lactate (pH 7.0) 2 g / L

Example 3 Preparation of Poly (3-hydroxybutyrate-co-lactate) (P (3HB-co-LA)) from Recombinant E. coli using glycerol as a carbon source

Simultaneously with pPs619C1337-CPPCT540 vector and pMCS104ReAB vector described in Examples 1-3 and LB agar plates containing 100 mg / L ampicillin and 34 mg / L chloramphenicol as antibiotics One transformed recombinant Escherichia coli XL1-Blue colony was inoculated into 3 mL of LB medium containing antibiotics such as 100 mg / L ampicillin and 34 mg / L chloramphenicol at a rate of 200 rpm at 30 ° C. Incubate for 24 hours with stirring. 1 mL of the culture was inoculated into 100 mL of MR medium containing 100 mg / L ampicillin and 34 mg / L chloramphenicol and 20 g / L or 50 g / L glycerol, which was then subjected to 200 rpm at 30 ° C. The main culture was started while incubating at a stirring speed. The culture was carried out for 4 days. In case of MR medium, the initial pH was adjusted to 7 using 10 N NaOH. The medium used for this culture is shown in Table 10 below.

After completion of the culture, cells were recovered from the culture by centrifugation. The recovered cells were washed three times with distilled water, and then dried in a drier at 100 ° C. for 24 hours. Some of the dried cells were collected and subjected to gas chromatography (GC) analysis to synthesize P (3HB-co-LA). ) Content was measured. Standards used in the analysis were P (3HB-co-3HV) copolymer (of which 3HV content was about 12% by weight) and polylactate homopolymer. GC analysis results are shown in Table 11 below. As a result of the analysis, it was confirmed that the P (3HB-co-LA) copolymer could be biosynthesized from glycerol. The P (3HB-co-LA) copolymer could be prepared from glycerol through the recombinant E. coli according to the present invention. there was.

Table 10

Table 11

badge	Early temperament	Biosynthetic Polymer Type	Polymer content (weight ratio)	LA content in the polymer (molar ratio)
MR	G2 ^*	P (3HB-LA)	53.08	64.75
MR	G2, NaL ⁺	P (3HB-LA)	51.38	69.92
MR	G5 ^**	P (3HB-LA)	40.11	53.13
MR	G5, NaL	P (3HB-LA)	53.37	51.94

* G2: Glycerol 20 g / L

** G5: Glycerol 50 g / L

+ NaL: Sodium lactate (pH 7.0) 2 g / L

Claims

Lactate polymers comprising genes of enzymes that convert lactate to lactyl-CoA and genes of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate, and can use glycerol as a substrate Or cells or plants having the ability to generate hydroxyalkanoate-lactate copolymers to lactate and glycerol; Or cultivated or grown in an environment containing lactate, glycerol and hydroxyalkanoate;

Recovering the lactate polymer or hydroxyalkanoate-lactate copolymer from the cell or plant

A process for preparing lactate polymers or hydroxyalkanoate-lactate copolymers from glycerol.
The method of claim 1, wherein the cell or plant does not include any one or more of a gene of an enzyme that converts lactate to lactyl-CoA and a gene of PHA synthase that uses lactyl-CoA as a substrate and uses glycerol as a substrate. And a cell or plant which is capable of being transformed with at least one of a gene of an enzyme for converting lactate to lactyl-CoA and a gene of PHA synthase using lactyl-CoA as a substrate.
The method of claim 1, wherein the hydroxyalkanoate in the hydroxyalkanoate-lactate copolymer is 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxy. 4-hydroxybutyrate, medium chain length (D) -3-hydroxycarboxylic acid having 6 to 14 carbon atoms, 2-hydroxypropionic acid ), 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid acid), 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid ( 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid hydroxyhexadecanoic acid, 4-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid ( 4-hydroxyoctanoic acid), 4-hydroxydecanoic acid, 5-hydroxyvaleric acid, 5-hydroxyhexanoic acid, 6-hydroxy degree 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- 3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7- Octenic acid (3-hydroxy-7-octenoic acid), 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid (3-hydroxy-9-de cenoic 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 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 3-hydroxy-6-methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid -5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3 -Hydroxy-6-methylno 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy 3-hydroxy-7-methyl-6-octenoic acid, malic acid, 3-hydroxysuccinic acid-methyl ester, 3- 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelanic acid methyl ester acid-methyl ester), 3-hydroxysebacic acid-methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxyseba Citrate-ethyl ester (3-hydroxysebacic) acid-ethyl ester, 3-hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzil ester, 3-hydroxy- 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyrate 3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyjade Phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxy valeric acid (para-cyanophenoxy -3-hydroxyvaleric acid), para-cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid (para-nitrophenoxy-3-) hydr oxyhexanoic acid), 3-hydroxy-5-phenylvaleric acid, 3-hydroxy-5-cyclohexylbutyric acid, 3,12- Dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy- 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-6,7-epoxydodecanoic acid, 3-hydroxy 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid -cyano-3-hydroxyheptanoic acid), 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid acid), 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy acid -8-claw 3-hydroxy-8-chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid 8-bromooctanoic acid), 3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 6- 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid (3 -hydroxy-2-methylvaleric acid), and 3-hydroxy-2,6-dimethyl-5-heptenoic acid.
The method of claim 1, wherein the gene of the enzyme for converting lactate to lactyl-CoA is propionyl-CoA transferase gene ( pct ).
The method of claim 1, wherein the gene of the enzyme converting lactate to lactyl-CoA is a pct gene derived from Clostridium propionicum .
According to claim 1, wherein the gene of the enzyme that converts lactate to lactyl-CoA

The nucleotide sequence of SEQ ID NO: 1;

A nucleotide sequence in which T78C, T669C, A1125G, and T1158C is mutated in the nucleotide sequence of SEQ ID NO: 1;

A nucleotide sequence of which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1;

A nucleotide sequence of which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and a Gly335Asp is mutated in the amino acid sequence of SEQ ID NO: 2;

A nucleotide sequence in which T669C, A1125G and T1158C is mutated in the nucleotide sequence of SEQ ID NO: 1, and Asp65Gly is mutated in the amino acid sequence of SEQ ID NO: 2;

A nucleotide sequence in which T669C, A1125G and T1158C is mutated in the nucleotide sequence of SEQ ID NO: 1, and Asp65Asn is mutated in the amino acid sequence of SEQ ID NO: 2;

A nucleotide sequence in which T669C, A1125G, and T1158C is mutated in the nucleotide sequence of SEQ ID NO: 1, and a Thr199Ile is mutated in the amino acid sequence of SEQ ID NO: 2;

A nucleotide sequence in which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and Ala243Thr is mutated in an amino acid sequence of SEQ ID NO: 2;

A nucleotide sequence in which A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and Asp257Asn is mutated in an amino acid sequence of SEQ ID NO: 2; And

And wherein T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala in the amino acid sequence of SEQ ID NO: 2 has a nucleotide sequence selected from the group consisting of mutated sequences.
The base of claim 6, wherein the gene of the enzyme converting lactate to lactyl-CoA is mutated in T78C, T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1, and Val193Ala is mutated in the amino acid sequence of SEQ ID NO: 2. 8. Having a sequence.
The method of claim 1, wherein the gene of PHA synthase using lactyl-CoA as a substrate is a PHA synthase gene of pseudomonas sp. 6-19 .
According to claim 1, wherein the gene of PHA synthase using the lactyl-CoA as a substrate

The amino acid sequence of SEQ ID NO: 4; or

Having a base sequence corresponding to an amino acid sequence comprising one or more mutations selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in the amino acid sequence of SEQ ID NO: 4 Way.
The method of claim 1, wherein the gene of the PHA synthase using the lactyl-CoA as a substrate has a base sequence corresponding to the amino acid sequence of E130D, S325T, S477G and Q481K in the amino acid sequence of SEQ ID NO: 4 .
The method of claim 1, wherein the cell or plant further comprises a gene of an enzyme that produces hydroxyacyl-CoA from glycerol.
The method of claim 11, wherein the enzyme that produces hydroxyacyl-CoA from glycerol is ketothiolase and acetoacetyl-CoA reductase.
The method of claim 12, wherein the ketothiolase and acetoacetyl-CoA reductase are from Ralstonia eutropha .
The method of claim 1, wherein the cell is a bacterium.
The method of claim 14, wherein the bacterium is Escherichia coli.