# Patent

## Title
Method for production of polylactate using recombinant microorganism

## Abstract

It is an object of the present invention to provide a method for efficiently producing a polylactate via microbial fermentation with the use of a sugar as a starting material.
The method is a method for producing a polylactate, which comprises the steps of:

    
    
        (1) culturing a recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of the following amino acid sequence (a) or (b) in a medium containing a carbon source:
        (a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s); or
        (b) an amino acid sequence derived from the protein specified in (a) by additional deletion or substitution of one or several amino acid(s) other than the amino acids at positions 130, 325, 477, and 481 or by insertion of one or several amino acid residue(s); and
        (2) collecting the polylactate from the culture product obtained in step (1).

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2009/068402 filed Oct. 27, 2009, claiming priority based on Japanese Patent Application No. 2008-276185, filed Oct. 27, 2008, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a method for producing a polylactate using a recombinant microorganism.
BACKGROUND ART
In view of global environmental issues, biologically derived polyesters have been gaining attention as biodegradable plastics that can be readily degraded in nature or as “green” plastics that can be synthesized from renewable carbon resources such as sugars or plant oils. In particular, polylactates can be obtained at relatively low cost. The melting points thereof are 170° C. or higher. Therefore, polylactates have sufficient heat resistance and thus can be shaped by melt molding. Accordingly, polylactates have been expected to be used as highly practical biodegradable polymers.
However, polylactate production has been conventionally carried out by neutralizing and purifying lactic acid produced by microorganisms, forming a dimeric cyclic compound (lactide), and carrying out polymerization as described in JP Patent Publication (Kokai) No. 2004-204464 A, which is problematic in terms of cost.
Hitherto, many microorganisms have been reported to have the ability to produce a polyester using a sugar as a carbon source (Non-Patent Document 1). A representative example of a biodegradable plastic produced by a microorganism is poly-3-hydroxybutyrate (polyhydroroxybutyrate (PHB)) consisting of 3-hydroxybutyrate (3βB) monomers. PHB is a thermoplastic polymer having a melting temperature of approximately 180° C. PHB has excellent melting processability as well as biodegradability, which is advantageous. At the same time, PHB is hard and fragile because of its high crystallinity. That is to say, it has poor shock resistance, which is problematic in terms of physical properties.
As a method for solving problems regarding physical properties of PHB, a method for producing a polyester copolymer consisting of 3HB and a different hydroxyalkanoate using a microorganism has been developed.
For example, Patent Document 1 discloses a method for producing a copolymer consisting of 3HB and 3-hydroxyvalerate (3HV). In addition, Patent Document 2 discloses a method for producing a copolymer consisting of 3HB and 3HV by allowing a microorganism of the genus Methylobacterium (Methylobacterium sp.), Paracoccus (Paracoccus sp.), Alcaligenes (Alcaligenes sp.), or Pseudomonas (Pseudomonas sp.) to come into contact with a primary alcohol having a carbon number of 3 to 7.
Such copolymer consisting of 3HB and 3HV has a higher degree of flexibility than PHB. In addition, it has been confirmed that an increase in the 3HV content in a polyester copolymer results in enhanced flexibility. In the above method for producing a copolymer consisting of 3HB and 3HV using a microorganism, the 3HV content in a polyester copolymer is regulated by adding, for example, propionic acid in the case of Patent Document 1 or propane-1-ol in the case of Patent Document 3 to a medium.
For instance, P(3HB-co-3HH), which is a two-component polyester copolymer consisting of 3HB and 3-hydroxyhexanoate (hereinafter abbreviated as “3HH”), and a method for producing the same are disclosed in Patent Documents 4 and 5. In the methods for producing a P(3HB-co-3HH) copolymer disclosed in these Patent Documents, fermentative production from a fatty acid such as oleic acid or fat and oil such as olive oil is carried out using Aeromonas caviae isolated from the soil. In addition, it has been reported that a recombinant strain is obtained by cloning the PHA synthase gene from A. caviae and introducing the cloned gene into Alcaligenes eutrophus for production of P(3HB-co-3HH) with the use of fatty acid as a carbon source and the recombinant strain (Patent Document 6).
In addition, in any case of the above methods for producing a polyester copolymer using a microorganism, it is necessary to use a polyhydroxyalkanoate synthase, which is an enzyme protein having activity of directly synthesizing a polymer. In addition, it has been attempted to modify such synthase so as to control the monomer unit mole fraction. For example, Patent Document 7 discloses a mutant enzyme capable of producing PHB with a high 3HB content, which is obtained by modifying the amino acid sequence of a polyhydroxyalkanoate synthase of a microorganism that has been identified as Pseudomonas sp. 61-3.
Meanwhile, a polyester copolymer consisting of a non-3-hydroxyalkanoate component serving as a monomer unit is expected to have physical properties differing from those of the above polyester copolymer. Patent Document 8 discloses a method for producing, as an example of such polyester copolymer comprising a non-3-hydroxyalkanoate component serving as a monomer unit, a polyester copolymer consisting of 3HB and lactate (LA) by culturing Ralstonia eutropha (previous name: Alcaligenes eutrophus) incorporating the nucleic acid encoding propionyl-CoA transferase of Clostridium propionicum with the addition of lactate to a medium. The document further discloses a method for producing a copolymer consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and lactate by culturing Escherichia coli incorporating the nucleic acid encoding C. propionium-derived propionyl-CoA transferase and the nucleic acid encoding Pseudomonas sp. 61-3-derived polyhydroxyalkanoate synthase with addition of lactate and decenoic acid to a medium.
In the above, a polyester copolymer consisting of 3-hydroxyalkanoate serving as a monomer unit, a polyester copolymer consisting of a non-3-hydroxyalkanoate component serving as a monomer unit, and a method for producing the same using a microorganism are described.
However, there have not been any reports of methods for efficiently producing polylactate via microbial fermentation with the use of sugar as a starting material.

    Non-Patent Document 1: “Biodegradable Plastic Handbook,” Biodegradable Plastics Society, 1995, pp. 178-197, (published by NTS Inc.)
    Patent Document 1: JP Patent Publication (Kokai) No. 57-150393 A (1982)
    Patent Document 2: JP Patent Publication (Kokai) No. 5-74492 A (1993)
    Patent Document 3: JP Patent Publication (Kokoku) No. 7-79705 B (1995)
    Patent Document 4: JP Patent Publication (Kokai) No. 5-93049 A (1993)
    Patent Document 5: JP Patent Publication (Kokai) No. 7-265065 A (1995)
    Patent Document 6: JP Patent Publication (Kokai) No. 10-108682 A (1998)
    Patent Document 7: WO2003/100055
    Patent Document 8: WO2006/126796


DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
It is an object of the present invention to provide a method for efficiently producing a polylactate via microbial fermentation with the use of a sugar as a starting material.
Means for Solving Problem
The present inventors found that a recombinant microorganism disclosed in Patent Document 7 into which the nucleic acid encoding the polyhydroxyalkanoate synthase mutant has been introduced can efficiently produce a polylactate directly from a sugar. This has led to the completion of the following inventions.
(1) A method for producing a polylactate, which comprises the steps of:
(1) culturing a recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of the following amino acid sequence (a) or (b) in a medium containing a carbon source:
(a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s); or
(b) an amino acid sequence derived from the protein specified in (a) by additional deletion or substitution of one or several amino acid(s) other than the amino acids at positions 130, 325, 477, and 481 or by insertion of one or several amino acid residue(s); and
(2) collecting the polylactate from the culture product obtained in step (1).
(2) The production method according to (1), wherein the amino acid sequence of the protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate is the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4 by deletion, substitution, or addition of one or several amino acid(s).

(3) The production method according to (1), wherein the amino acid sequence of the protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate is the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 by deletion, substitution, or addition of one or several amino acid(s).

(4) The production method according to (1), wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of amino acids at positions 325 and 481 with different amino acids.

(5) The production method according to (1), wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of Ser at position 325 with Thr and Gln at position 481 with Lys.

(6) The production method according to (1), wherein any one of the proteins is a protein encoded by a recombinant expression vector introduced into a microorganism.

(7) A recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of the following amino acid sequence (a) or (b):

(a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s); or
(b) an amino acid sequence derived from the protein specified in (a) by additional deletion or substitution of one or several amino acid(s) other than the amino acids at positions 130, 325, 477, and 481 or by insertion of one or several amino acid residue(s).
(8) The recombinant microorganism according to (7) wherein the amino acid sequence of the protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate is the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4 by deletion, substitution, or addition of one or several amino acid(s).

(9) The recombinant microorganism according to (7), wherein the amino acid sequence of the protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate is the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 by deletion, substitution, or addition of one or several amino acid(s).

(10) The recombinant microorganism according to (7), wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of amino acids at positions 325 and 481 with different amino acids.

(11) The recombinant microorganism according to (7), wherein the amino acid sequence of a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of Ser at position 325 with Thr and Gln at position 481 with Lys.

(12) The recombinant microorganism according to (7), into which a recombinant expression vector having a gene encoding any one of the proteins is introduced.

This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2008-276185, which is a priority document of the present application.
Effects of the Invention
According to the production method of the present invention, a polylactate can be efficiently produced using an inexpensive carbon source as a starting material, and thus the production cost of a biodegradable plastic can be reduced.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically shows the configuration of a recombinant plasmid pTV118NPCTC1(ST/QK). In the figure, “phaC1(ST/QK)” denotes the STQK gene, “PCT” denotes the M. elsdenii-derived PCT gene, “PRe” denotes an R. eutropha-derived promoter, and “Plac” denotes an Escherichia coli lactose operon promoter.
FIG. 2 shows a molecular weight distribution curve for the polymer prepared in the Example.
FIG. 3A is a chart showing GC/MS analysis results for the polymer prepared using a recombinant plasmid pTV118NPCTC1(ST/QK) having the M. elsdenii-derived PCT gene and the phaC1(ST/QK) gene.
FIG. 3B is a chart showing GC/MS analysis results for the polymer prepared using a recombinant plasmid pTV118NPCTC1(WT) having The M. elsdenii-derived PCT gene and the phaC1(WT) gene.
FIG. 3C is a chart showing GC/MS analysis results for the polymer prepared using a PLA authentic preparation (MW20,000).
FIG. 3D is a chart showing GC/MS analysis results for the polymer prepared using a recombinant plasmid pTV118NPCTC1(ST/QK) having the M. elsdenii-derived PCT gene or the C. propionicum-derived PCT gene and the phaC1(ST/QK) gene.
FIG. 4 is a chart showing H-NMR spectral analysis results for the polymer prepared in the Example.
FIG. 5 schematically shows the configuration of a recombinant plasmid pTV118NPCTC1(WT). In the figure, “phaC1(WT)” denotes Pseudomonas sp. 61-3-derived polyhydroxyalkanoate synthase gene (wild type), “PCT” denotes the M. elsdenii-derived PCT gene, “PRe” denotes an Alcaligenes eutrophus-derived promoter, and “Plac” denotes a lactose operon promoter.
FIG. 6 is a graph showing time-dependent changes in lactoyl CoA production in accordance with different PCT genes.
FIG. 7 is a graph showing a comparison in terms of polylactate productivity between the M. elsdenii-derived PCT gene and the C. propionicum-derived PCT gene.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention relates to a method for producing a polylactate, which comprises the steps of:
(1) culturing a recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of the following amino acid sequence (a) or (b) in a medium containing a carbon source:
(a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s); or
(b) an amino acid sequence derived from the protein specified in (a) by additional deletion or substitution of one or several amino acid(s) other than the amino acids at positions 130, 325, 477, and 481 or by insertion of one or several amino acid residue(s); and
(2) collecting the polylactate from the culture product obtained in step (1). Hereinafter, proteins and recombinant microorganisms used in the present invention and conditions for the production method of the present invention are described.
[1] A Protein Capable of Catalyzing a Reaction of Transferring CoA to Propionic Acid and/or Lactate (LA)
A “protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or LA” used in the present invention is a protein having activity of catalyzing a reaction of transferring CoA from an appropriate CoA substrate to propionic acid and/or LA. The protein having such activity is generally referred to as propionyl-CoA transferase (PCT). Hereinafter, the protein is denoted by “PCT” in the present invention.
Table 1 shows representative examples of PCT origins (microorganism names) that have been reported in the past and information on references disclosing the nucleotide sequences encoding the microorganisms.








TABLE 1





Microorganism name
Reference information







Clostridium propionicum
Eur. J. Biochem., 2002, Vol. 269, pp. 372-380


Megasphaera elsdenii
U.S. Pat. No. 7,186,541


Staphylococcus aureus
Eur. J. Biochem., 2002, Vol. 269, pp. 372-380


Escherichia coli
Eur. J. Biochem., 2002, Vol. 269, pp. 372-380









In addition to the above examples shown in table 1, any PCT that has been reported in the past can be used in the present invention. In addition, as long as a “protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or LA” is used, even a protein consisting of an amino acid sequence derived from the amino acid sequence of a known PCT by deletion, substitution, or addition of one or several amino acid(s) can be used. Here, the term “more amino acids” used in connection with the PCT amino acid sequence refers to 1 to 50 amino acids, preferably 1 to 25 amino acids, and more preferably 10 or less amino acids.
Catalytic activity in a reaction of transferring CoA to propionic acid and/or LA can be determined according to, for example, the method described by A. E. Hofmeister et al. (Eur. J. Biochem., vol. 206, pp. 547-552).
A preferable example of PCT in the present invention is Megasphaera elsdenii-derived PCT. The amino acid sequence thereof is shown in SEQ ID NO: 4. An example of the nucleotide sequence of nucleic acid (DNA) encoding the amino acid sequence is shown in SEQ ID NO: 3.
Another example of a preferable PCT in the present invention is Staphylococcus aureus-derived PCT. The amino acid sequence of Staphylococcus aureus-derived PCT is shown in SEQ ID NO: 6. An example of the nucleotide sequence of a nucleic acid (DNA) encoding the amino acid sequence is shown in SEQ ID NO: 5. As described in the Examples below, Staphylococcus aureus-derived PCT is superior to Megasphaera elsdenii-derived PCT in terms of lactate CoA productivity in the early phase of microbial culture. This suggests that lactate CoA and a polylactate can be more rapidly produced. Accordingly, the use of Staphylococcus aureus-derived PCT is advantageous in that the cost of polylactate production can be reduced.
In the present invention, any of the above types of PCTs may be used alone, or PCTs from a plurality of species may be used in combination. For instance, Megasphaera elsdenii-derived PCT is superior to Staphylococcus aureus-derived PCT in terms of lactateCoA productivity in the late phase of microbial culture. It is thought that the combined use of both PCTs allows maintenance of lactate CoA productivity for a longer time period than previously possible, based on such characteristics of Megasphaera elsdenii-derived PCT and the aforementioned characteristics of Staphylococcus aureus-derived PCT.
[2] A Protein Capable of Catalyzing a Reaction of Polyhydroxyalkanoate Synthesis
The protein capable of catalyzing polyhydroxyalkanoate synthesis of the present invention is a protein consisting of:
(a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of amino acids at positions 130, 325, 477, and 481 with different amino acid(s); or
(b) an amino acid sequence derived from the protein specified in (a) by additional deletion or substitution of one or several amino acid(s) other than the amino acids at positions 130, 325, 477, and 481 or by insertion of one or several amino acid residue(s). A protein defined in (a) is obtained by partially mutating the amino acid sequence of Pseudomonas species (Pseudomonas sp.) 61-3-derived polyhydroxyalkanoate synthase described in Patent Document 7. A protein defined in (b) is obtained by additionally mutating a protein defined in (a) to an extent such that the activity of the protein defined in (a) can be maintained. In addition, the term “more amino acids” used in connection with the protein defined in (b) refers to 1 to 50 amino acids, preferably 1 to 25 amino acids, and more preferably 10 or fewer amino acids. Hereinafter, a protein capable of catalyzing polyhydroxyalkanoate synthesis of the present invention is referred to as “PhaCm.” In addition, Patent Document 7 is incorporated herein by reference in its entirety.
Preferable examples of PhaCm include a single mutant protein obtained by substitution of any one of the amino acids at positions 130, 325, 477, and 481 of the amino acid sequence shown in SEQ ID NO: 2 listed in table 6 or 7 in Patent Document 7, a double mutant protein obtained by substitution of any two of the same, a triple mutant protein obtained by substitution of any three of the same, and a quadruple mutant protein obtained by substitution of all four of the same.
A preferable protein is a double mutant protein obtained by substitution of any two of the above. A particularly preferable protein is a double mutant protein obtained by substitution of Ser at position 325 with Thr and Gln at position 481 with Lys (hereinafter abbreviated as “STQK”).
DNA encoding PhaCm can be produced via recombination by a site-specific mutagenesis method known to persons skilled in the art based on the amino acid sequence (SEQ ID NO: 2) of a polyhydroxyalkanoate synthase from Pseudomonas species (Pseudomonas sp.) 61-3 and the nucleotide sequence (SEQ ID NO: 1) of DNA encoding the same. In addition, as described in Patent Document 7, the PhaCm activity of catalyzing polyhydroxyalkanoate synthesis can be confirmed by obtaining a host cell via transformation with the above nucleic acid capable of expressing PhaCm and examining the polyhydroxyalkanoate accumulation capacity of the host cell.
[3] Nucleic Acids Encoding the Proteins
The nucleic acids encoding proteins (1) and (2) described above are introduced into microorganisms, followed by protein transcription and translation therein. The thus obtained proteins are preferably used. Each nucleic acid to be introduced into a microorganism is preferably incorporated into a vector.
A vector for introducing the aforementioned nucleic acid into a microorganism may be a vector that can autonomously replicate in a host. Preferable examples thereof include plasmid DNA and phage DNA. Examples of a vector for introducing a nucleic acid into Escherichia coli include: plasmid DNAs such as pBR322, pUC18, and pBLuescriptII; and phage DNAs such as EMBL3, M13, and λgtII. In addition, examples of a vector used for introduction into yeast include YEp13 and YCp50.
In addition, examples of a vector used for introduction of a nucleic acid into a microorganism of the genus Ralstonia or Pseudomonas include pLA2917(ATCC37355) having an RK2 replication origin and pJRD215 (ATCC 37533) having an RSF1010 replication origin, which can be replicated/conserved in a wide range of hosts.
The nucleic acids (preferably DNAs) encoding proteins (1) and (2) described above can be inserted into vectors by a gene recombination technique known to persons skilled in the art. In addition, upon recombination, it is preferable to ligate DNA (inserted into a vector) downstream of a promoter that can control transcription/translation of a relevant protein from the DNA. Any promoter can be used as long as it can control gene transcription in a host. For instance, when Escherichia coli is used as a host, a trp promoter, a lac promoter, a PL promoter, a PR promoter, a T7 promoter, or the like can be used. In addition, when yeast is used as a host, a gal1 promoter, a gal10 promoter, or the like can be used. Further, when a microorganism of the genus Pseudomonas is used as the microorganism of the present invention, a region such as the region that is supposed to contain a promoter located upstream of the phaC1Ps gene or the phbCRe operon can be used as a promoter.
In addition, if necessary, the vector of the present invention can be ligated to a terminator sequence, an enhancer sequence, a splicing signal sequence, a polyA addition signal sequence, a ribosome binding sequence (SD sequence), and a selection marker gene, which can be used in a microorganism into which a nucleic acid is introduced. Examples of a selection marker gene include: a gene involved in intracellular biosynthesis of a nutrient such as an amino acid or a nucleic acid and a gene encoding a fluorescent protein such as luciferase, in addition to a drug-resistant gene such as an ampicillin-resistant gene, a tetracycline-resistant gene, a neomycin-resistant gene, a kanamycin-resistant gene, or a chloramphenicol-resistant gene.
Any aforementioned nucleic acid, which is preferably incorporated into a vector, is introduced into a microorganism by a method known to persons skilled in the art. Examples of a method for recombination of a vector into a microorganism include a calcium phosphate method, an electroporation method, a spheroplast method, a lithium acetate method, a conjugational transfer method, and a method using calcium ions.
[4] Microorganism
The recombinant microorganism of the present invention is a microorganism expressing proteins (1) and (2) described above, and it is preferably a microorganism transformed via introduction of the nucleic acids capable of functionally expressing proteins (1) and (2). Preferable examples of such microorganism include bacteria of the genus Pseudomonas such as the Pseudomonas sp. 61-3 strain, bacteria of the genus Ralstonia such as R. eutropha, bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Corynebacterium, yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, and yeasts of the genus Candida such as Candida maltosa. Of these, Escherichia coli, bacteria of the genus Corynebacterium, and R. eutropha are preferable. Escherichia coli and bacteria of the genus Corynebacterium are particularly preferable.
In the case of a microorganism such as R. eutropha, which originally contains a unique polyhydroxyalkanoate synthase, a microorganism lacking the capacity to express such unique polyhydroxyalkanoate synthase is preferably used. Such microorganism lacking expression capacity can be produced by treating a microorganism with a chemical mutation source such as nitrosoguanidine or a physical mutation source such as UV radiation, introducing a mutant nucleic acid (obtained by modifying a nucleic acid encoding a polyhydroxyalkanoate synthase so as not to cause the functional expression of the enzyme) into a microorganism, or inducing “homologous recombination.” Destruction of the polyhydroxyalkanoate synthase gene can be confirmed by examining whether a hybridizing band shifts to the expected position when compared with a wild-type strain-derived band upon Southern hybridization with the use of a portion of the gene as a probe.
[5] Production of a Polylactate
A polylactate can be produced by culturing a recombinant microorganism into which any aforementioned nucleic acid has been introduced in a medium containing a carbon source, causing generation and accumulation of a polylactate in cultured microbial cells or a culture product, and collecting the polylactate from the cultured microbial cells or the culture product.
Preferably, each recombinant microorganism of the present invention is cultured under general culture conditions for the microorganism depending on the recombinant microorganism type, except for the medium composition.
A medium with a specific composition is not particularly required herein. However, it is preferable to use a medium with the limited content of a nitrogen source (other than a carbon source), an inorganic salt, or a different organic nutrient. An example of a medium for culturing a recombinant microorganism obtained by incorporating a nucleic acid into a bacterium of the genus Ralstonia or Pseudomonas is a medium with the nitrogen source content limited to 0.01% to 0.1%.
Examples of a carbon source include carbohydrates such as glucose, fructose, sucrose, and maltose. In addition, a fat-and-oil-related substance with a carbon number of 4 or higher can be used as a carbon source. Examples of a fat-and-oil-related substance with a carbon number of 4 or higher include: natural fat and oil such as corn oil, soybean oil, safflower oil, sunflower oil, olive oil, coconut oil, palm oil, rapeseed oil, fish oil, whale oil, lard, or beef tallow; fatty acid such as butanoic acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, or myristic acid, or an ester of such fatty acid, and alcohol such as octanol, lauryl alcohol, oleyl alcohol, or palmityl alcohol, or an ester of such alcohol.
Examples of a nitrogen source include peptone, meat extract, yeast extract, and corn steep liquor, in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate. Examples of an inorganic substance include primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
Preferably, culture is carried out under aerobic conditions via general shake culture or the like at 25° C. to 37° C. within 24 hours or longer after transcriptional expression of proteins [1] and [2] described above. During culture, an antibiotic such as kanamycin, ampicillin, or tetracycline may be added to a medium. If all or some of the DNAs encoding proteins [1] and [2] are ligated under the regulation of an inducible promoter, a factor that induces transcription of the promoter may be added to a medium.
In one preferable embodiment of the present invention, the method for producing a polylactate comprises culturing recombinant Escherichia coli into which an expression vector containing the nucleic acid encoding M. elsdenii-derived PCT (SEQ ID NO: 3) or the nucleic acid encoding S. aureus-derived PCT (SEQ ID NO: 5) and the nucleic acid encoding STQK has been introduced. According to the method of the present invention, a polylactate can be produced from inexpensive blackstrap molasses without the addition of a monomer component (e.g., LA) that constitutes a polymer of interest to a medium. This is advantageous in terms of production cost.
In the present invention, a polylactate can be collected by a method for collecting a polylactate or PHA from a microorganism that is known to persons skilled in the art. For example, microbial cells are collected from a culture solution via centrifugation, followed by washing and drying. Then, the dried microbial cells are suspended in chloroform and heated for extraction of a polyester of interest in a chloroform fraction. Further, methanol is added to the resulting chloroform solution for precipitation of a polylactate. The supernatant is removed via filtration or centrifugation, followed by drying. Thus, the purified polylactate can be obtained.
It is possible to confirm whether the collected polyester is a polylactate by a general method such as gas chromatography or a nuclear magnetic resonance method.
The present invention is described below in more detail with reference to the Examples in a non-limited manner. In addition, experimental operations used in the Examples were conducted according to manuals introducing experimental operations (e.g., Sambrook et al., Molecular cloning: a laboratory manual, 2nd ed. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and instructions included with a variety of reagents and kits.
EXAMPLES
Example 1
(1) Production of a Recombinant Microorganism
Genomic DNA was extracted from M. elsdenii (ATCC17753) with the use of a DNeasy Tissue Kit (Qiagen). Subsequently, primer DNA of a forward primer comprising the EcoRI recognition sequence and that of a reverse primer comprising the PstI recognition sequence were synthesized in order to amplify a nucleic acid encoding propionyl CoA transferase (accession No. J04987) by PCR.
PCR reaction was performed for 1 cycle of 94° C. for 2 minutes and 30 cycles of 94° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 2 minutes in a reaction solution containing KOD-Plus-DNA polymerase (1 U), PCR buffer, 1 mM MgSO4, the primers (15 pmol each), and 0.2 mM dNTPs (each produced by TOYOBO Co., Ltd.) by means of iCycler (BioRad) with the use of the genomic DNA as a template. Then, an amplified fragment with a size of approximately 1,500 by was collected, followed by digestion with EcoRI and PstI. Thus, a DNA fragment was obtained.
In addition, DNA fragments each encoding propionyl CoA transferase were obtained from S. aureus (ATCC10832) and C. propionicum (ATCC25522) in the manner described above. The following are PCR amplification conditions used herein: a PCR reaction (enzyme KOD-PLUS) of 1 cycle of 94° C. for 1 minute, 30 cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 2 minutes, and 94° C. for 2 minutes.
The following are the sequences for the primer sets used for amplification of individual genes:







M. elsdenii PCT:


MePCTN:


5′-atgagaaaagtagaaatcattac-3′


 


MePCTC:


5′-ttattttttcagtcccatgggaccgtcctg-3′


 


C. propionicum PCT:


CpPCTN:


5′-gggggccatgggaaaggttcccattattaccgcagatgag -3′


 


CpPCTC:


5′-ggggggctcgagtcaggacttcatttccttcagacccat-3′


 


S. aureus PCT:


SpctN:


5′-gtgccatggaacaaatcacatggcacgac-3′


 


SpctC:


5′-cacgaattcatactttatgaattgattg-3′






Plasmid pTV118N (Takara Shuzo Co., Ltd.) was digested with EcoRI and PstI, followed by dephosphorylation with alkaline phosphatase. Subsequently, ligation was performed with the addition of the M. elsdenii-derived DNA fragment. Thus, a recombinant plasmid PTV118N M.E.-PCT with a size of approximately 4.7 kbp containing DNA encoding propionyl CoA transferase was prepared.
Similarly, the C. propionicum-derived and S. aureus-derived PCT genes are separately introduced into the NcoI-BamHI site and the NcoI-EcoRI site of a pTV118N vector (Takara Shuzo Co., Ltd.). Thus, PTV118N C.P.-PCT and PTV118N S.A.-PCT expression plasmids were produced.
According to the method described by Takase et al. (J. Biochem., 2003, vol. 133, pp. 139-145), a plasmid pGEMC1(ST/QK)AB containing DNAs encoding STQK was prepared.
pGEMC1(ST/QK)AB was digested with BamHI to collect a DNA fragment with a size of approximately 6 kbp. T4 polymerase (200 units) was allowed to act at 37° C. for 5 minutes in a 3 mM Tris acetate buffer solution (pH 7.9) containing 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, 0.1 mg/mL BSA, and 0.1 mM dNTP. Thus, a DNA fragment encoding STQK was obtained.
pTV118N M.E.-PCT was digested with PstI. T4 polymerase was allowed to act under the conditions described above, followed by dephosphorylation with alkaline phosphatase. The above DNA fragment encoding phaCm was subjected to ligation. DNA encoding STQK was introduced to the SalI site of pTV118N M.E.-PCT. Thus, plasmid pTV118N-PCT-C1(ST/QK)AB (approximately 9.6 kbp) was obtained. pTV118N-PCT-C1 was obtained by amplifying the region excluding phaA and phaB of pTV118N-PCT-C1AB by PCR, followed by self-ligation of the amplified product (FIG. 1). Escherichia sp. W3110 competent cells were transformed using pTV118N-PCT-C1(ST/QK).
In addition, pTV118N-C.P PCT-C1(STQK) having the DNA fragment encoding phaCm was obtained using the PTV118N C.P.-PCT expression plasmid in the manner described above, followed by transformation of Escherichia sp. W3110 competent cells.
(2) Polymer Production
For culture of the obtained transformant, active colonies formed on the LB agar medium were inoculated onto an LB liquid medium (10 ml) contained in a 100-ml Erlenmeyer flask and subjected to shake culture at 30° C. until OD 0.6 to 1.0 was reached (IFM (130 rpm); Oriental Giken Inc.). The preculture solution of grown bacterial cells was inoculated onto a medium for main culture (200 ml) contained in a 500-ml Erlenmeyer flask so that it accounted for 1% of the resultant, followed by shake culture.
After culture in the 200-ml medium by the method described above, bacterial cells were collected from the obtained culture solution using a 50-ml Falcon tube (3000×g, 5 min, RT), followed by overnight freezing at −80° C. The frozen bacterial cells were dried by a lyophilizer (LABCONCO, Model 77400) for 2 days.
The dried bacterial cells were placed in a pressure-proof test tube. Chloroform was added thereto in an amount of 1 ml per 100 mg of bacterial cells, followed by reflux in a water bath at 95° C. for 3 hours. The sample subjected to reflux was cooled to room temperature, filtered with a 0.22-Mm PTFE filter, and introduced into a silicone centrifugal tube for removal of bacterial cells. The extracted sample was dried at room temperature until chloroform was completely removed therefrom. Hexane (2 ml) was added to the pellet obtained after drying, followed by vortexing for approximately 1 minute and centrifugation (6,000×g, 15 minutes, RT) for removal of the supernatant. Washing with hexane was carried out twice in such manner. The dried sample was dissolved in chloroform (2 ml). The resultant was placed in a glass vial and dried. The resultant was designated as a polymer analysis sample.
(3) Polymer Analysis
(i) GPC
Chloroform (1 mL) was added to the polymer (approximately 1 mg) collected in (2) above, followed by filtration with a 0.2-μm PTFE filter (ADVANTEC). The resulting solution was used as a sample and subjected to determination by GPC under the conditions described below.
System: Shimadzu Prominence GPC System
Column: TSKgel-Super THZ-M (6.0 mm×150 mm)
Eluent: CHCl3 
Flow rate: 0.8 mL/minute
Temperature: 40° C.
Detection: 10A refractive index detector
Sample amount: 10 μL
FIG. 2 shows a distribution curve of determined molecular weights. A molecular weight calibration curve was created with the use of a standard polystyrene. The molecular weight was represented by a value converted to the standard polystyrene molecular weight. As a result, the polymer molecular weight (mW) was 22,000.

(ii) GC/MS Analysis

After culture in the 200-ml medium by the method described above, bacterial cells were collected from the obtained culture solution using a 50-ml Falcon tube (3000×g, 5 min, RT), followed by overnight freezing at −80° C. The frozen bacterial cells were dried by a lyophilizer (LABCONCO, Model 77400) for 2 days. Then, 100 mg of the dried bacterial cells were weighed and placed in a pressure-proof test tube. Chloroform (1.6 ml) was added thereto and the resultant was allowed to stand still overnight at room temperature. A methanol-sulfuric acid mixture (a liquid mixture; methanol:sulfuric acid=17:3) (1.6 ml) was added to the bacterial cell/chloroform solution. An internal standard (100 μl) (benzoic acid: 10 mg/CHCl3: 10 ml) was added thereto, followed by reflux in a water bath at 95° C. for 3 hours for methylation. After the end of methylation, the sample was cooled to room temperature and placed in a disposable test tube (φ: 18 mm). Milli-Q water (800 μl) was added thereto, followed by stirring for approximately 30 seconds. Thereafter, the resultant was allowed to stand still until it was separated into an aqueous layer and a solvent layer. After separation, the chloroform layer was sampled with a Pasteur pipette, filtered with a 0.22-μm PTFE filter, and introduced into a 2-ml vial for analysis.
Methylation of a polylactate authentic preparation was carried out in the following manner. First, PLA-0020 (Wako Pure Chemical Industries, Ltd.; weight-average molecular weight: 20,000) (20 mg) was dissolved in chloroform (10 ml) in a 20-ml vial. A portion of the resultant (1 ml) was placed in a pressure-proof test tube. Chloroform (0.6 ml), methanol sulfuric acid (1.6 ml), and an internal standard (100 μl) were added thereto, followed by reflux in a water bath at 95° C. for 3 hours. After the end of methylation, the standard sample was cooled to room temperature and placed in a disposable test tube (φ: 18 mm). Milli-Q water (800 μl) was added thereto and the resultant was stirred for approximately 30 seconds. The resultant was allowed to stand still until it was separated into an aqueous layer and a solvent layer. After separation, a chloroform layer was introduced into a 2-ml vial with the use of a Pasteur pipette, and the resultant was used for analysis.
GC/MS analysis was performed under the conditions described below.
The methylated product was analyzed using a GC/MS (HP6890 Series GC system/5973 Mass SelectiveDetector) equipped with an Agilent DB-1 column (122-10G3; 150 meters×0.25 mm×1 mm) or an Agilent DB-1 column (122-1063; 60 meters×0.25 mm×1 mm). The analysis methods implemented with the use of the columns are described below.
122-10G3: Holdup at 150° C. for 2 minutes; temperature increase at a rate of 5° C./1 minute; and holdup at 300° C. for 10 minutes
122-1063: Holdup at 120° C. for 5 minutes; temperature increase at a rate of 5° C./1 minute; and holdup at 300° C. for 10 minutes
FIGS. 3A to 3D each show analysis results obtained under the above conditions and the MS spectrum for methyl lactate. Note that, only the results shown in FIG. 3D were obtained from the sample from which no chloroform had been removed. As a result of GC/MS, the polymer collected in (2) was confirmed to contain LA as a monomer unit.
(iii) NMR Analysis
A sample was prepared by dissolving the polymer collected in (2) in deuterated chloroform, followed by 1H-NMR (FIG. 4) determination at 300 MHz. As a result, it was found that the polymer collected in (2) contained lactate as a monomer unit.
(4) PCT Expression and Lactoyl CoA Synthesis in Microorganisms
The PTV118N M.E.-PCT, PTV118N C.P.-PCT, and PTV118N S.A.-PCT recombinant plasmids produced in (1) were used for transformation of Escherichia sp. W3110 competent cells.
After preculture, each cell line was inoculated onto a 200-ml LB medium contained in a 2-L flask so as to result in a content of 2%, followed by culture at 37° C. at 180 rpm for 3 hours. Expression was induced using 10 mM IPTG at approximately OD600=0.5, followed by culture at 30° C. at 80 rpm for 6 hours. Next, bacterial cells were collected via centrifugation, followed by culture at 37° C. in M9 (+1.5% glucose, 10 mM MgSO4, 10 mM calcium pantothenate) (OD=20; 3 ml), followed by sampling in an appropriate manner.
Bacterial cells (approximately 1×105 cells) were collected for sample preparation (n=3). The sample was applied to a suction filter system and washed twice with Milli-Q water. A filter was placed (face down) in a petri dish containing an MeOH solution (2 ml) and left at room temperature for 10 minutes. Then, the MeOH solution (1.6 ml) was introduced into a centrifugal tube. Chloroform (1.6 ml) and Milli-Q water (640 ul) were mixed together and suspended, followed by centrifugation at 4600 g at 4° C. for 5 min. The water+MeOH layer (1.5 ml) was subjected to centrifugal filtration using a 5 k ultrafiltration membrane (Millopore) for approximately 2 hours. The filtrate was collected and lyophilized. The resultant was concentrated 200-fold and dissolved in Milli-Q water containing a secondary internal standard substance, followed by CE-MS analysis. CE-MS analysis conditions were determined in accordance with Anal. Chem. 2002, 74, 6224-6229, “Pressure-Assisted Capillary Electrophoresis Electrospray Ionization Mass Spectrometry for Analysis of Multivalent Anions.” FIG. 6 shows the results.
Based on FIG. 6, it has been revealed that the S. aureus-derived PCT gene exhibits lactate CoA productivity at a level greater than that of the M. elsdenii-derived PCT gene in the early phase of culture.
(5) Influence of the PCT Gene upon Polylactate Productivity
FIG. 7 shows quantification results for a lactate derivative obtained by GC/MS analysis in (ii) above.
Based on the results shown in FIG. 7, it has been revealed that M. elsdenii-derived PCT is superior to C. propionicum-derived PCT in terms of favorable contribution to polylactate productivity.
Comparative Example
An expression vector was prepared by replacing the nucleotide sequence encoding STQK contained in the pTV118N-PCT-C1(ST/QK) plasmid produced in Example 1 with the nucleotide sequence encoding the protein described below. FIG. 5 shows an outline of the construction of the expression vector.
pTV118N-PCT-C1(WT): Polyhydroxyalkanoate synthase of Pseudomonas sp. 61-3 (wild type; SEQ ID NO: 1)
Escherichia coli W3110 competent cells were transformed using pTV118N-PCT-C1. For culture of the obtained transformant, active colonies formed on the LB agar medium were inoculated onto an LB liquid medium (10 ml) contained in a 100-ml Erlenmeyer flask, followed by shake culture at 30° C. to result in OD 0.6 to 1.0 (IFM (130 rpm); Oriental Giken Inc.). The grown precultured cell solution was inoculated to a content of 1% on a medium for main culture (200 ml) contained in a 500-ml Erlenmeyer flask, followed by shake culture.
After culture in the 200-ml medium by the method described above, bacterial cells were collected from the obtained culture solution using a 50-ml Falcon tube (3000×g, 5 min, RT), followed by overnight freezing at −80° C. The frozen bacterial cells were dried by a lyophilizer (LABCONCO, Model 77400) for 2 days. Then, 100 mg of the dried bacterial cells were weighed and introduced into a pressure-proof test tube. Chloroform (1.6 ml) was added thereto and the resultant was allowed to stand still overnight at room temperature. Methanol sulfuric acid (1.6 ml) and an internal standard (100 μl) (benzoic acid: 10 mg/CHCl3: 10 ml) were added to the bacterial cells/chloroform solution. The resultant was subjected to reflux in a water bath at 95° C. for 3 hours for methylation. After the end of methylation, the sample was cooled to room temperature and introduced into a disposable test tube (φ: 18 mm), Milli-Q water (800 μl) was added thereto and the resultant was stirred for approximately 30 seconds. The resultant was allowed to stand still until it was separated into an aqueous layer and a solvent layer. After separation, a chloroform layer was collected using a Pasteur pipette and filtered with a 0.22-μm PTFE filter into a 2-ml vial, followed by GS/MS analysis.
GS/MS analysis was performed in the manner described in Example 1. Based on the results shown in FIG. 3B, it was confirmed that substantially no polymer containing lactate can be produced using pTV118NPCTC1(WT).
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A method for producing a polylactate, which comprises the steps of:
(1) culturing a recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate consisting of the amino acid sequence shown in SEQ ID NO: 4 or 6 and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of
an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s); and
(2) collecting the polylactate from the culture product obtained in step (1).

2. The production method according to claim 1, wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of amino acids at positions 325 and 481 with different amino acids.

3. The production method according to claim 1, wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of Ser at position 325 with Thr and Gln at position 481 with Lys.

4. The production method according to claim 1, wherein any one of the proteins is a protein encoded by a recombinant expression vector introduced into a microorganism.

5. A recombinant microorganism having a protein capable of catalyzing a reaction of transferring CoA to propionic acid and/or lactate consisting of the amino acid sequence shown in SEQ ID NO: 4 or 6 and a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis consisting of an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of at least one of the amino acids at positions 130, 325, 477, and 481 with different amino acid(s).

6. The recombinant microorganism according to claim 5, wherein the amino acid sequence of the protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of amino acids at positions 325 and 481 with different amino acids.

7. The recombinant microorganism according to claim 5, wherein the amino acid sequence of a protein capable of catalyzing a reaction of polyhydroxyalkanoate synthesis is an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution of Ser at position 325 with Thr and Gln at position 481 with Lys.

8. The recombinant microorganism according to claim 5, into which a recombinant expression vector having a gene encoding any one of the proteins is introduced.