KR101260187B1 - Recombinant microorganism able to produce polylactate or polylactate copolymer from glycerol and method for producing polylactate or polylactate copolymer from glycerol using the same - Google Patents

Abstract

The present invention relates to microorganisms capable of producing lactate polymers or copolymers using glycerol and to methods of preparing lactate polymers or copolymers using glycerol as one of the substrates using such microorganisms. According to the present invention, a gene of an enzyme for converting lactate to lactyl-CoA and a gene of polyhydroxyalkanoate (PHA) synthetase using lactyl-CoA as a substrate are described. When introduced and cultured with microorganisms using glycerol as a substrate, lactate polymers and copolymers can be efficiently produced.

Description

Recombinant microorganism able to produce polylactate or polylactate copolymer from glycerol and method for producing polylactate or polylactate copolymer from glycerol using the same}

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, 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.

We believe Clostridium to provide lactyl-CoA A variant of polyhydroxyalkanoate synthase of Pseudomonas sp. 6-19 using propionyl-CoA transferase derived from propionicum and lactyl-CoA produced thereby as a substrate It was possible to successfully synthesize the lactate polymer and copolymer (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 which does not contain any one or more of the gene of the enzyme and (b) the gene of PHA synthase using lactyl-CoA as a substrate is transformed into one or more genes of (a) and (b) That is obtained by transforming a cell or plant that does not have the genes (a) and (b) with the genes (a) and (b), or (b) without the gene (a) Cells or plants can be obtained by transforming them with the gene (a), but are not limited thereto, and for example, the genes in the cells having any one of the above (a) and Amplification and Owl It was transformed with other genes within the scope of the invention.

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 used as lactyl-CoA. The gene of the enzyme that converts to Clostridium propionicum derived propionyl-CoA transferase gene. 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 more we preferred it to Clostridium CpPct540 gene using glycerol produce lactate polymer or copolymer It is a gene of propionicum- derived propionyl-CoA transferase variant.

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 nucleotide 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 Ralstonia Preference is given to those derived from 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 cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment 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 Escherichia 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 or 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 effecting 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 if 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 transformations were described by Sambrook et. al ., this can be readily accomplished using the calcium chloride method described in section 1.82 of 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. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

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. 6-19 (KCTC 11027BP) to transmission of the CoA from the polymer polymerase derived phaC1 _{Ps6 -19} 337, acetyl -CoA lactate, an enzyme which was converted to the riktil -CoA Clostridium propionicum-derived propionyl -CoA trans peora Kinase (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).

Also, 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 Clostridium propionicum derived from Clostridium propionicum from the propionyl-CoA transferase gene (CPPCT) genes are 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 . 6-19 derived PHA  Substrate Specificity of Synthetase transition Sieve production

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). Results of studies on mutants suitable for short-chain-length PHA (SCL-PHA) production have been reported (Takase et al. al ., Biomacromolecules , 5: 480, 2004). Based on this, the inventors have made phaC1 _{Ps6 -19} synthase variants using SDM method for amino acids affecting SCL activity (Domestic Patent Application No. 10-2006-0116234).

More specifically, it was prepared as follows. Pseudomonas sp. 6-19 (KCTC 11027BP) Pseudomonas sp to isolate derived PHA synthase (phaC1 _{Ps6 -19)} gene. 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 reaction resulted in 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.

pSYL105 vector: In (Lee et al, Biotech Bioeng, 1994, 44... 1337-1347) Ralstonia DNA fragments containing PHB producing operon derived from eutropha H16 were cut with BamHI / EcoRI and inserted into the BamHI / EcoRI recognition site of pBluescript II (Stratagene) to prepare a pReCAB recombinant vector.

The pReCAB vector is known to express PHA synthase (phaC _RE ) and monomer feeders (phaA _RE & phaB _RE ) at all times by the PHB operon promoter and to work well in E. coli (Lee et. al ., Biotech . Bioeng ., 1994, 44: 1337-1347. pReCAB vector cleaved with BstBI / SbfI to R. eutropha H16, remove the PHA synthase (phaC _RE) and then by inserting the phaC1 _{Ps6 -19} gene obtained above in the BstBI / SbfI recognition site to prepare a recombinant vector pPs619C1-ReAB.

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 conversion of amino acids 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'-aac ggg agg gaa cct gca gg-3 '

Nucleotide sequence of the phaC1 _{Ps6 -19} gene of the produced by-pPs619C1 ReAB recombinant vector and confirmed by sequencing, showed in SEQ ID NO: 3, exhibited the amino acid sequence encoded thereby in SEQ ID NO: 4.

The phaC1 _{Ps6 -19} to confirm the PHB synthesis of whether synthase pPs619C1-ReAB recombinant vector was transformed into E. coli XL-1Blue (Stratagene) , 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 by amino acid sequence analysis, and the phaC1 _{Ps6 -19} synthase variants shown in Table 1 using the SDM method using the primers of SEQ ID NOs: 13 to 18 Made them.

Recombinant vector Nucleic Acid Substitution Amino acid substitutions primer pPs619C1200-ReAB AGC → ACC S325T SEQ ID NO: 13/14 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'-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'-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 '

These recombinant vectors were transformed into E. coli XL1-Blue, and grown in PHB detection medium (LB agar, glucose 20g / L, Nile red 0.5μg / 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 produced 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 is Clostridium The fragment obtained by PCR of the chromosomal DNA of propionicum using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 was used. At this time, the Nde I site originally present in the wild type CP-PCT was used by SDM method for easy cloning. Removed.

SEQ ID NO: 19'5-ggaattcATGAGAAAGGTTCCCATTATTACCGCAGATGA-3 '

SEQ ID NO: 20'-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg-3 '

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'-gcc cat atg tct aga tta gga ctt cat ttc c-3 '

phaC1 _{Ps6 -19} synthase SCL mutant of phaC1 _Ps6 by cutting a _-19 300 a pPs619C1300-ReAB vector containing the Sbf I / Nde I Ralstonia eutrophus After removing the H16-derived monomer feed enzymes (phaA _RE & phaB _RE ), 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 5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc-3 '

SEQ ID NO: 24'-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: 5'-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-3'

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'-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: 5'-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 '

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

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

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

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> Clostridium propionicum  Derived Propionyl - CoA Transferase  Mutant Library Construction and Screening

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. cp - to introduce the random mutations in pct pPs619C1300-CPPCT (The Republic of Korea Patent Application No. 10-2006-0116234) by the mold, and SEQ ID NO: 39, and Mn ^{2 +} using primers of 40 is added 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 was cut containing a mutant of phaC1 _{Ps6 -19} 300 pPs619C1300-CPPCT vector with Sbf I / Nde I wild-type cp - After removal of the 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.

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.

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 a 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> pPs619C1337 - CPPCT540  Production of 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 detection 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> pMCS104ReAB Production

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). pSYL105 (Lee et al . Biotechnol . Bioeng . 44: 1337, 1994), p10499A (Park et al ., FEMS ), wherein the phaAB gene obtained by cleaving with PstI was cleaved with PstI. Microbiol . Lett ., 214: 217, 2002) to prepare p10499PhaAB. 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> As a carbon source  Polylactate from Recombinant Escherichia Coli Using Glycerol PLA ) Produce

One recombinant E. 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.

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.

badge Early temperament Biosynthetic
Polymer type Polymer content
(Weight) LA content in polymers
(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> As a carbon source  Poly (3- from Recombinant Escherichia Coli Using Glycerol Hydroxybutyrate - co - Lactate ) (P (3 HB - co - LA )) Produce

Simultaneously with the pPs619C1337-CPPCT540 vector and pMCS104ReAB vector described in <Example 1-3> and <Example 1-4> on 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.

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.

badge Early temperament Biosynthetic
Polymer type Polymer content
(Weight) LA content in polymers
(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

<110> LG CHEM, LTD. <120> Recombinant microorganism able to produce polylactate or          polylactate copolymer from sucrose and method for producing          polylactate or polylactate copolymer from sucrose using the same <130> 2009-0492 <150> KR10-2009-0124171 <151> 2009-12-14 <160> 47 <170> Kopatentin 1.71 <210> 1 <211> 1572 <212> DNA <213> Clostridium propionicum propionyl-CoA transferase <400> 1 atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60 acagttacaa caagtggttt cgttggaaat gcaatccctg aggctcttga tagagctgta 120 gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta cctatgttta ttgtggttct 180 caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240 tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300 atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360 cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420 ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480 caggaatatt tattctaccc tgcttttcct attcatgtag ctcttattcg tggtacttac 540 gctgatgaaa gcggaaatat cacatttgag aaagaagttg ctcctctgga aggaacttca 600 gtatgccagg ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140 tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttattgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cc 1572 <210> 2 <211> 524 <212> PRT <213> Clostridium propionicum propionyl-CoA transferase <400> 2 Met Arg Lys Val Pro Ile Ile Thr Ala Asp Glu Ala Ala Lys Leu Ile   1 5 10 15 Lys Asp Gly Asp Thr Val Thr Thr Ser Gly Phe Val Gly Asn Ala Ile              20 25 30 Pro Glu Ala Leu Asp Arg Ala Val Glu Lys Arg Phe Leu Glu Thr Gly          35 40 45 Glu Pro Lys Asn Ile Thr Tyr Val Tyr Cys Gly Ser Gln Gly Asn Arg      50 55 60 Asp Gly Arg Gly Ala Glu His Phe Ala His Glu Gly Leu Leu Lys Arg  65 70 75 80 Tyr Ile Ala Gly His Trp Ala Thr Val Pro Ala Leu Gly Lys Met Ala                  85 90 95 Met Glu Asn Lys Met Glu Ala Tyr Asn Val Ser Gln Gly Ala Leu Cys             100 105 110 His Leu Phe Arg Asp Ile Ala Ser His Lys Pro Gly Val Phe Thr Lys         115 120 125 Val Gly Ile Gly Thr Phe Ile Asp Pro Arg Asn Gly Gly Gly Lys Val     130 135 140 Asn Asp Ile Thr Lys Glu Asp Ile Val Glu Leu Val Glu Ile Lys Gly 145 150 155 160 Gln Glu Tyr Leu Phe Tyr Pro Ala Phe Pro Ile His Val Ala Leu Ile                 165 170 175 Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Phe Glu Lys Glu             180 185 190 Val Ala Pro Leu Glu Gly Thr Ser Val Cys Gln Ala Val Lys Asn Ser         195 200 205 Gly Gly Ile Val Val Val Gln Val Glu Arg Val Val Lys Ala Gly Thr     210 215 220 Leu Asp Pro Arg His Val Lys Val Pro Gly Ile Tyr Val Asp Tyr Val 225 230 235 240 Val Val Ala Asp Pro Glu Asp His Gln Gln Ser Leu Asp Cys Glu Tyr                 245 250 255 Asp Pro Ala Leu Ser Gly Glu His Arg Arg Pro Glu Val Val Gly Glu             260 265 270 Pro Leu Pro Leu Ser Ala Lys Lys Val Ile Gly Arg Arg Gly Ala Ile         275 280 285 Glu Leu Glu Lys Asp Val Ala Val Asn Leu Gly Val Gly Ala Pro Glu     290 295 300 Tyr Val Ala Ser Val Ala Asp Glu Glu Gly Ile Val Asp Phe Met Thr 305 310 315 320 Leu Thr Ala Glu Ser Gly Ala Ile Gly Gly Val Pro Ala Gly Gly Val                 325 330 335 Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr             340 345 350 Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly         355 360 365 Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly     370 375 380 Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr 385 390 395 400 Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val                 405 410 415 Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys             420 425 430 Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala         435 440 445 Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe     450 455 460 Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile 465 470 475 480 Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile                 485 490 495 Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe             500 505 510 Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser         515 520 <210> 3 <211> 1677 <212> DNA <213> PHA synthase <400> 3 atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360 gggcacttcg tgatcaacct catgaccgaa gcgatggcgc cgaccaacac cgcggccaac 420 ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720 cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag 780 gaacagcgag agtggggcct gtcgacctac atcgaagccc tcaaggaagc ggttgacgtc 840 gttaccgcga tcaccggcag caaagacgtg aacatgctcg gggcctgctc cggcggcatc 900 acttgcactg cgctgctggg ccattacgcg gcgattggcg aaaacaaggt caacgccctg 960 accttgctgg tgagcgtgct tgataccacc ctcgacagcg acgtcgccct gttcgtcaat 1020 gaacagaccc ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080 gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc 1140 aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg gaacaacgac 1200 accacacggt tgcccgcggc gttccacggc gacctgatcg aactgttcaa aaataaccca 1260 ctgattcgcc cgaatgcact ggaagtgtgc ggcaccccca tcgacctcaa gcaggtgacg 1320 gccgacatct tttccctggc cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380 aagtcggcgc aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcag cgggcatatc 1440 cagagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg 1500 gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc atacagattc ctggtggctg 1560 cactggcagg cctggcaggc ccaacgctcg ggcgagctga aaaagtcccc gacaaaactg 1620 ggcagcaagg cgtatccggc aggtgaagcg gcgccaggca cgtacgtgca cgaacgg 1677 <210> 4 <211> 559 <212> PRT <213> PHA synthase <400> 4 Met Ser Asn Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn   1 5 10 15 Thr Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu              20 25 30 Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His          35 40 45 Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu      50 55 60 Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Asp Arg Arg Phe Ala  65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr                  85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Asp Glu Ser Asn             100 105 110 Leu Ala Pro Lys Asp Val Ala Arg Gly His Phe Val Ile Asn Leu Met         115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val     130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val                 165 170 175 Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr Glu Gly             180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro         195 200 205 Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu Leu Val Val Pro Pro Gln     210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg                 245 250 255 Asn Pro Thr Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu             260 265 270 Ala Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys         275 280 285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala     290 295 300 Leu Leu Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Asp Val Ala                 325 330 335 Leu Phe Val Asn Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr             340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp         355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu     370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe                 405 410 415 Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly Thr             420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser Leu Ala Gly         435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln     450 455 460 Leu Phe Gly Gly Asn Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr                 485 490 495 Ser Thr Glu Val Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr             500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln         515 520 525 Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala     530 535 540 Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 atgcccggag ccggttcgaa 20 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 cgttactctt gttactcatg atttgattgt ctctc 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 gtacgtgcac gaacggtgac gcttgcatga gtg 33 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 aacgggaggg aacctgcagg 20 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 ctgaccttgc tggtgaccgt gcttgatacc acc 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 ggtggtatca agcacggtca ccagcaaggt cag 33 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 cgagcagcgg gcatatcatg agcatcctga acccgc 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 gcgggttcag gatgctcatg atatgcccgc tgctcg 36 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 atcaacctca tgaccgatgc gatggcgccg acc 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 ggtcggcgcc atcgcatcgg tcatgaggtt gat 33 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 ggaattcatg agaaaggttc ccattattac cgcagatga 39 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 gctctagatt aggacttcat ttccttcaga cccattaagc cttctg 46 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 aggcctgcag gcggataaca atttcacaca gg 32 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 gcccatatgt ctagattagg acttcatttc c 31 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaattcgtgc tgtcgagccg cgggcatatc 30 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gatatgcccg cggctcgaca gcacgaattc 30 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 gaattcgtgc tgtcgagcca tgggcatatc 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gatatgccca tggctcgaca gcacgaattc 30 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gaattcgtgc tgtcgagctt tgggcatatc 30 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gatatgccca aagctcgaca gcacgaattc 30 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 gaattcgtgc tgtcgagcta tgggcatatc 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gatatgccca tagctcgaca gcacgaattc 30 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gaattcgtgc tgtcgagcgg cgggcatatc 30 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gatatgcccg ccgctcgaca gcacgaattc 30 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gggcatatca aaagcatcct gaacccgc 28 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gcgggttcag gatgcttttg atatgccc 28 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gggcatatca tgagcatcct gaacccgc 28 <210> 36 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gcgggttcag gatgctcatg atatgccc 28 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gggcatatcc gcagcatcct gaacccgc 28 <210> 38 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 gcgggttcag gatgctgcgg atatgccc 28 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 cgccggcagg cctgcagg 18 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 ggcaggtcag cccatatgtc 20 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 ttcgtgctgt cgagcagagg gcatatc 27 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 gatatgccct ctgctcgaca gcacgaa 27 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 agacatatgc aagtaccttg ccgacatcta tg 32 <210> 44 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gatgacaacg tcagtcatga tttgattgtc tctctg 36 <210> 45 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 cagagagaca atcaaatcat gactgacgtt gtcatc 36 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 gcaggtcagc ccatatgcag 20 <210> 47 <211> 1677 <212> DNA <213> Artificial Sequence <220> <223> pPs619C1-335-540 (1537bp-3213bp, direct) <400> 47 atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360 gggcacttcg tgatcaacct catgaccgat gcgatggcgc cgaccaacac cgcggccaac 420 ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720 cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag 780 gaacagcgag agtggggcct gtcgacctac atcgaagccc tcaaggaagc ggttgacgtc 840 gttaccgcga tcaccggcag caaagacgtg aacatgctcg gggcctgctc cggcggcatc 900 acttgcactg cgctgctggg ccattacgcg gcgattggcg aaaacaaggt caacgccctg 960 accttgctgg tgaccgtgct tgataccacc ctcgacagcg acgtcgccct gttcgtcaat 1020 gaacagaccc ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080 gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc 1140 aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg gaacaacgac 1200 accacacggt tgcccgcggc gttccacggc gacatgatcg aactgttcaa aaataaccca 1260 ctgattcgcc cgaatgcact ggaagtgtgc ggcaccccca tcgacctcaa gcaggtgacg 1320 gccgacatct tttccctggc cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380 aagtcggcgc aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcgg cgggcatatc 1440 atgagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg 1500 gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc atacagattc ctggtggctg 1560 cactggcagg cctggcaggc ccaacgctcg ggcgagctga aaaagtcccc gacaaaactg 1620 ggcagcaagg cgtatccggc aggtgaagcg gcgccaggca cgtacgtgca cgaacgg 1677

Claims (15)

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 for converting the lactate to lactyl-CoA is Clostridium propionicum ( Clostridium propionicum ) derived pct gene.
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. Having a sequence.
According to claim 1, wherein the gene of the PHA synthase using the lactyl-CoA as a substrate is pseudomonas genus 6-19 ( 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 variations 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.

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