Patent Grant
8980615
This application is a National Stage of International Application No. PCT/JP2011/057077 filed Mar. 24, 2011, claiming priority based on Japanese Patent Application No. 2010-069688 filed Mar. 25, 2010, the contents of all of which are incorporated herein by reference in their entirety.
The present invention relates to a recombinant microorganism to which desired functions are imparted by introducing a predetermined gene into a host microorganism and a method for producing aliphatic polyester using the same.
Aliphatic polyester is attracting attention as biodegradable plastic that can be easily degraded in nature or “green” plastic that can be synthesized from recyclable carbon resources such as sugar or vegetable oil. Currently, as aliphatic polyester, polyester having a lactic acid backbone, such as polylactic acid, is practically used.
As a technology for producing aliphatic polyester such as polylactic acid using a recombinant microorganism, for example, the technology disclosed in Patent Document 1 (WO 2006/126796) is known. Patent Document 1 discloses recombinant Escherichia coli prepared by introducing a gene encoding an enzyme that converts lactic acid to lactic-acid CoA and a gene encoding an enzyme that synthesizes polyhydroxyalkanoate using lactic-acid CoA as a substrate into host Escherichia coli. According to the technology disclosed in Patent Document 1, a Clostridium propionicum-derived pct gene is used as a gene encoding an enzyme that converts lactic acid to lactic-acid CoA. Furthermore, according to this technology, a Pseudomonas sp. 61-3 strain-derived phaC2 gene is used as a gene encoding an enzyme that synthesizes polyhydroxyalkanoate using lactic-acid CoA as a substrate.
However, Patent Document 1 has problems in that the productivity of aliphatic polyester such as polylactic acid cannot be said to be sufficient, and various examinations for improvement of the productivity are insufficient. For example, Patent Document 2 (WO 2008/062999) discloses an attempt to enhance the capacity of synthesizing a lactic acid homopolymer or a polylactic acid copolymer using lactic-acid CoA as a substrate through introduction of a specific mutation into a phaC1 gene from the Pseudomonas sp. 6-19 strain.
The above technology for producing aliphatic polyester such as polylactic acid using a recombinant microorganism involves accumulating aliphatic polyester within the microorganism. Hence, target aliphatic polyester is recovered by disrupting the microorganism.
However, conventionally, such technology for producing aliphatic polyester such as polylactic acid using a recombinant microorganism has been problematic in that productivity is low since aliphatic polyester is accumulated within the microorganism, and complicated steps are required in order to disrupt the microorganism and then recovering aliphatic polyester. Hence, an object of the present invention is to provide a recombinant microorganism having good aliphatic polyester productivity and to provide a method for producing aliphatic polyester using the recombinant microorganism.
As a result of intensive studies to achieve the above object, the present inventors have discovered that, in a recombinant microorganism prepared by introducing a propionyl CoA transferase gene and a polyhydroxyalkanoate synthase gene from a predetermined microorganism, aliphatic polyester such as polylactic acid is produced extracellularly, and thus they have completed the present invention.
Specifically, the present invention encompasses the following (1) to (11).
(1) A method for producing aliphatic polyester, comprising culturing a recombinant microorganism prepared by introducing a gene encoding a protein that has activity of converting lactic acid to lactic-acid CoA and a gene encoding a protein that has activity of synthesizing polyhydroxyalkanoate using hydroxyacyl CoA as a substrate into a host microorganism, and then recovering aliphatic polyester from medium.
(2) The method for producing aliphatic polyester according to (1), wherein the aliphatic polyester comprises oligomers that are mainly a dimer, a trimer, a tetramer, and a pentamer.
(3) The method for producing aliphatic polyester according to (1), wherein the aliphatic polyester has the lactic acid backbone.
(4) The method for producing aliphatic polyester according to (1), wherein the aliphatic polyester is polylactic acid.
(5) The method for producing aliphatic polyester according to (1), wherein the medium is a minimal medium.
(6) The method for producing aliphatic polyester according to (1), wherein the recombinant microorganism is cultured for 48 hours or more and then the aliphatic polyester is recovered.
(7) The method for producing aliphatic polyester according to (1), wherein the gene encoding a protein that has activity of synthesizing polyhydroxyalkanoate using the hydroxyacyl CoA as a substrate is at least one gene selected from an Alcanivorax borkumensis-derived gene, a Hyphomonas neptunium-derived gene, a Rhodobacter sphaeroides-derived gene, a Rhizobium etli-derived gene, a Pseudomonas sp.-derived gene, and a Haloarcula marismortui-derived gene.
(8) The method for producing aliphatic polyester according to (1), wherein the gene encoding the protein that has activity of synthesizing polyhydroxyalkanoate using hydroxyacyl CoA as a substrate is the following gene (a), (b), or (c):
(a) a gene encoding a protein that comprises the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18;
(b) a gene encoding a protein that comprises an amino acid sequence having a substitution, a deletion, or an addition of 1 or a plurality of amino acids with respect to the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18, and has the above activity; or
(c) a gene hybridizing under stringent conditions to a polynucleotide that has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, or 17, and encoding a protein that has the above activity;
(9) A recombinant microorganism, which is prepared by introducing:
a gene encoding a protein having activity of converting lactic acid to lactic-acid CoA; and
one or more genes encoding a protein(s) having activity of synthesizing polyhydroxyalkanoate using hydroxyacyl CoA as a substrate, which is selected from an Alcanivorax borkumensis-derived gene, a Hyphomonas neptunium-derived gene, a Rhodobacter sphaeroides-derived gene, a Rhizobium etli-derived gene, a Pseudomonas sp.-derived gene, and a Haloarcula marismortui-derived gene that are a gene encoding a protein and, into a host microorganism.
(10) The recombinant microorganism according to (9), wherein the gene that encodes a protein having activity of synthesizing polyhydroxyalkanoate using hydroxyacyl CoA as a substrate is the following gene (a), (b), or (c):
(a) a gene encoding a protein that comprises the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18;
(b) a gene encoding a protein that comprises an amino acid sequence having a substitution, a deletion, or an addition of 1 or a plurality of amino acids with respect to the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18, and has the above activity; or
(c) a gene hybridizing under stringent conditions to a polynucleotide that has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, or 17 and encoding a protein having the above activity.
(11) The recombinant microorganism according to (9), wherein the host microorganism is Escherichia coli.
This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2010-069688, which is a priority document of the present application.
According to the present invention, a recombinant microorganism capable of producing aliphatic polyester extracellularly can be provided. Specifically, the recombinant microorganism according to the present invention has higher aliphatic polyester productivity than conventional recombinant microorganisms. Also, through the use of the recombinant microorganism according to the present invention, a method for producing aliphatic polyester with high productivity can be provided.
Hereinafter, the recombinant microorganism and the method for producing aliphatic polyester using the same according to the present invention are as described in detail.
The recombinant microorganism according to the present invention is prepared by introducing a propionyl CoA transferase gene (pct gene) and a predetermined polyhydroxyalkanoate synthase gene into a host microorganism, and it produces aliphatic polyester outside the host microorganism. In addition, the term “aliphatic polyester” as used herein refers to not only polymers (macromolecular substances) each having a molecular weight of several thousands to several tens of thousands, but also oligomers each having 2 to 5 monomeric units (that is, dimer to pentamer).
Propionyl CoA Transferase Gene
In the present invention, a propionyl CoA transferase gene (hereinafter, referred to as “pct gene”) is not particularly limited and any gene can be used herein, as long as it encodes a protein having activity of converting lactic acid to lactic-acid CoA. Specifically, as a pct gene, any gene that encodes a protein having propionyl CoA transferase activity can be used. The term “propionyl CoA transferase activity” refers to activity of catalyzing a reaction by which CoA is transferred to propionic acid. Specifically, activity of catalyzing a reaction by which CoA is transferred from an appropriate CoA substrate to propionic acid is referred to as propionyl CoA transferase activity. The propionyl CoA transferase can transfer CoA not only to propionic acid, but also to lactic acid from a CoA substrate.
Table 1 shows representative examples of origins (names of microorganisms) of pct genes reported to date and document information disclosing the information of nucleotide sequences encoded by the genes.
Clostridium propionicum
Megasphaera elsdenii
Staphylococcus aureus
Escherichia coll
In the present invention, any pct gene that has been reported to date can be used in addition to those listed in Table 1 above. Also, any protein comprising an amino acid sequence that has a deletion, a substitution, or an addition of 1 or several amino acids with respect to a known amino acid sequence of a pct protein can be used, as long as it has propionyl CoA transferase activity. In addition, the term “several” used in relation to the amino acid sequence of a pct protein refers to 1 to 50, preferably 1 to 25, and more preferably 10 or less amino acids. Catalytic activity exhibited by propionyl CoA transferase can be measured according to a method described by A. E. Hofineister et al., (Eur. J. Biochem., Vol. 206, pp. 547-552), for example.
Examples of the pct gene include a Megasphaera elsdenii-derived gene and a Staphylococcus aureus-derived gene. The nucleotide sequence of the coding region in the Megasphaera elsdenii-derived pct gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the pct gene is shown in SEQ ID NO: 2. Also, the nucleotide sequence of the coding region in the Staphylococcus aureus-derived pct gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the pct gene is shown in SEQ ID NO: 4. The protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 has propionyl CoA transferase activity, and particularly activity of synthesizing lactic-acid CoA using lactic acid as a substrate.
Also, in the present invention, examples of the pct gene is not limited to the gene having the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or 4, and may be a pct gene encoding a protein that comprises an amino acid sequence having a deletion, a substitution, or an addition of 1 or a plurality of amino acid sequences with respect to the relevant amino acid sequence, and has activity of converting lactic acid to lactic-acid CoA. Here, the term “a plurality of amino acids” refers to, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1 to 3 amino acids.
Furthermore, in the present invention, the pct gene may be a pct gene encoding a protein that comprises an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence similarity with respect to the amino acid sequence shown in SEQ ID NO: 2 or 4, and has activity of converting lactic acid to lactic-acid CoA. Here, the value of sequence similarity refers to a value that is found using a computer program for blast algorithm implementation, database storing gene sequence information, and default setting.
Furthermore, in the present invention, the pct gene may also be a pct gene that comprises a polynucleotide hybridizing under stringent conditions to at least a portion of a gene having the nucleotide sequence shown in SEQ ID NO: 1 or 3, and, encodes a protein having activity of converting lactic acid to lactic-acid CoA. Here the term “stringent conditions” refers to conditions wherein namely a specific hybrid is formed, but no non-specific hybrid is formed. Examples thereof include hybridization at 45° C. with 6×SSC (sodium chloride/sodium citrate), followed by washing at 50° C. to 65° C. with 0.2 to 1×SSC and 0.1% SDS. Alternatively, examples of such conditions further include conditions of hybridization at 65° C. to 70° C. with 1×SSC, followed by washing at 65° C. to 70° C. with 0.3×SSC. Hybridization can be performed by a conventionally known method such as a method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
In addition, deletion, substitution, or addition of an amino acid(s) can be performed by modifying a nucleotide sequence encoding the above transcription factor by a technique known in the art. Mutation can be introduced into a nucleotide sequence by a known technique such as a Kunkel method or a Gapped duplex method or a method according thereto. For example, mutation is introduced using a mutagenesis kit (e.g., Mutant-K and Mutant-G (both are trade names, TAKARA Bio)) or the like using site-directed mutagenesis, or a LA PCR in vitro Mutagenesis Series Kit (trade name, TAKARA Bio). Furthermore, a mutagenesis method may be a method using a chemical agent for mutation represented by EMS (ethyl methanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N′-nitro-N nitrosoguanidine, other carcinogenic compounds or the like, or a method using radiation processing as represented by X-ray processing, alpha ray processing, beta ray processing, gamma ray processing, or ion beam processing, or ultraviolet [UV] treatment.
Polyhydroxyalkanoate Synthase Gene
In the present invention, as a polyhydroxyalkanoate synthase gene (also referred to as a PHA synthase gene), at least one gene selected from an Alcanivorax borkumensis-derived gene, a Hyphomonas neptunium-derived gene, a Rhodobacter sphaeroides-derived gene, a Rhizobium etli-derived gene, a Pseudomonas sp.-derived gene, and a Haloarcula marismortui-derived gene is used. In particular, as a PHA synthase gene, an Alcanivorax borkumensis-derived gene and/or a Hyphomonas neptunium-derived gene is preferably used. In addition, the term “PHA synthase gene” refers to a gene encoding a protein that has activity of synthesizing polyhydroxyalkanoate using hydroxyacyl CoA as a substrate.
As the Alcanivorax borkumensis-derived gene, the PHA synthase gene derived from the SK2 strain preserved in the ATCC under Accession Number: 700651 can be preferably used. Also, as the Hyphomonas neptuniums-derived gene, a PHA synthase gene derived from the strain preserved in the NBRC under Accession Number: 14232 is preferably used.
As the Rhodobacter sphaeroides-derived gene, a PHA synthase gene derived from the strain preserved in the ATCC (American Type Culture Collection) under Accession Number: BAA-808D is preferably used. As the Rhizobium etli-derived gene, a PHA synthase gene derived from the CFN strain preserved in the NBRC (NITE Biological Resource Center) under Accession Number: 15573 is preferably used. As the Pseudomonas sp.-derived gene, a PHA synthase gene derived from the 61-3 strain preserved in the JCM (Japan Collection of Microorganisms) under Accession Number: 10015 is preferably used. As the Haloarcula marismortui-derived gene, a PHA synthase gene from the strain preserved in the JCM under Accession Number: 8966 is preferably used.
Specifically, the nucleotide sequence of the coding region in the Alcanivorax borkumensis (ATCC 700651)-derived PHA synthase gene is shown in SEQ ID NO: 5, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 6. The nucleotide sequence of the coding region in the Hyphomonas neptunium (NBRC 14232)-derived PHA synthase gene is shown in SEQ ID NO: 7, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 8.
Also, examples of the Rhodobacter sphaeroides (BAA-808D)-derived gene include the PHA synthase gene specified by Accession Number: YP354337 and the PHA synthase gene specified by Accession Number: ABA79557. The nucleotide sequence of the coding region in the PHA synthase gene specified by Accession Number:YP354337 is shown in SEQ ID NO: 9, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 10. The nucleotide sequence of the coding region in the PHA synthase gene specified by Accession Number: ABA79557 is shown in SEQ ID NO: 11, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 12.
The nucleotide sequence of the coding region in the Rhizobium etli CFN strain-derived PHA synthase gene is shown in SEQ ID NO: 13, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 14. The nucleotide sequence of the coding region in the Pseudomonas sp. 61-3 strain-derived PHA synthase gene is shown in SEQ ID NO: 15, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 16. The nucleotide sequence of the coding region in the Haloarcula marismortui (JCM 8966)-derived PHA synthase gene is shown in SEQ ID NO: 17, and the amino acid sequence of the protein to be encoded by the gene is shown in SEQ ID NO: 18.
Furthermore, in the present invention, examples of the PHA synthase gene are not limited to those having the nucleotide sequences encoding the amino acid sequences specified by the above specific SEQ ID NOS. The PHA synthase gene may be a PHA synthase gene encoding a protein that comprises an amino acid sequence having a deletion, a substitution, or an addition of 1 or a plurality of amino acid sequences with respect to the relevant amino acid sequence, and, has activity of synthesizing polylactic acid using lactic-acid CoA as a substrate. Here, the term “a plurality of amino acids” refers to, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1 to 3 amino acids.
Furthermore, in the present invention, the PHA synthase gene may be a PHA synthase gene encoding a protein that comprises an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence similarity with the amino acid sequence specified by the above specific SEQ ID NO, and has activity of synthesizing polylactic acid using lactic-acid CoA as a substrate. Here, the value for sequence similarity refers to a value that is found by a computer program for blast algorithm implementation using database storing gene sequence information and the default setting.
Furthermore, in the present invention, the PHA synthase gene may be a PHA synthase gene comprising a polynucleotide that hybridizes under stringent conditions to at least a portion of a gene having the nucleotide sequence specified by the above specific SEQ ID NO:, and, encoding a protein having activity of synthesizing polylactic acid using lactic-acid CoA as a substrate. In addition, the term “stringent conditions” is synonymous with the conditions as described in the section of “Propionyl CoA transferase gene.”
Also, techniques described in the section of “Propionyl CoA transferase gene” can be applied for deletion, substitution, or addition of an amino acid(s).
In particular, the recombinant microorganism according to the present invention is prepared by introducing the above-described PHA synthase gene, so that it can produce an aliphatic polyester oligomer, and particularly, a lactic acid oligomer outside the microorganism. Here, an oligomer having the degree of polymerization that differs depending on the types of PHA synthase gene to be used herein can be produced. With the recombinant microorganism prepared by introducing the Alcanivorax borkumensis-derived PHA synthase gene, tetrameric and pentameric aliphatic polyester oligomers (e.g., lactic acid oligomers) can be produced. Also, with the recombinant microorganism prepared by introducing the Hyphomonas neptunium-derived PHA synthase gene, a tetrameric aliphatic polyester oligomer (e.g., a lactic acid oligomer) can be produced.
Host Microorganism
Examples of a host microorganism to be used in the present invention 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, yeast of the genus Saccharomyces such as Saccharomyces cerevisiae, and yeast of the genus Candida such as Candida maltosa. As a host microorganism, Escherichia coli is particularly preferably used.
A vector for introducing the above gene into a host cell may be a vector that is autonomously replicable in the host, and is preferably in the form of plasmid DNA or phage DNA. Examples of such a vector to be introduced into Escherichia coli include plasmid DNA such as pBR322, pUC18, and pBluescript II and phage DNA such as EMBL3, M13, and λgtII. Examples of a vector to be introduced into yeast include YEp13 and YCp50.
Both or either one of the above genes can be inserted into a vector by a gene recombination technique known by persons skilled in the art. Also, upon recombination, the above gene is preferably ligated downstream of a promoter capable of regulating transcription. As a promoter, any promoter capable of regulating transcription of a gene in a host can also be used herein. For example, 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 is used. When yeast is used as a host, a gall promoter, a gal10 promoter, or the like can be used.
Also, if necessary, a terminator sequence, an enhancer sequence, a splicing signal sequence, a polyA addition signal sequence, a ribosome binding sequence (SD sequence), a selection marker gene, and the like, which can be used in a microorganism for gene introduction, can be ligated to a vector. Examples of a selection marker gene include, in addition to drug resistance genes such as an ampicillin resistance gene, a tetracycline resistance gene, a neomycin resistance gene, a kanamycin resistance gene, and a chloramphenicol resistance gene, genes involved in intracellular biosynthesis of nutrients, such as amino acids or nucleic acids, or genes encoding fluorescent proteins such as green fluorescent protein.
The above vector can be introduced into a microorganism by a method known by persons skilled in the art. Examples of such a method for introducing a vector into a microorganism include a calcium phosphate method, electroporation, a spheroplast method, a lithium acetate method, a conjugal transfer method, and a method using calcium ions.
Production of Aliphatic Polyester
A target aliphatic polyester oligomer can be produced by culturing a recombinant microorganism (obtained by introducing the above pct gene and PHA synthase gene into a host microorganism) in medium containing carbon sources, causing generation and accumulation of the aliphatic polyester oligomer in the culture product, and then recovering the aliphatic polyester oligomer. The recombinant microorganism synthesizes lactic acid from sugar through a sugar metabolic pathway, and then propionyl CoA transferase encoded by the pct gene converts lactic acid into lactic acid-CoA. Furthermore, in the recombinant microorganism, PHA synthase encoded by the PHA synthase gene synthesizes an aliphatic polyester oligomer comprising lactic acid as a constitutional unit using lactic-acid CoA as a substrate. The oligomer may be polylactic acid (homopolymer) comprising only lactic acid as a constitutional unit, or a lactic acid-based copolymer comprising lactic acid and hydroxyalkanoic acid other than lactic acid as constitutional units. Also, oligomers to be produced in medium are mainly dimers, trimers, tetramers, and pentamers. Here, the term “mainly” means that the above oligomers account for 50% or more, preferably 70% or more, and more preferably 90% or more of the aliphatic polyester components contained in the medium.
When polylactic acid (homopolymer) is synthesized, hydroxyalkanoic acid other than lactic acid is not added to the medium, or a biosynthetic pathway for hydroxyalkanoic acid other than lactic acid in the host microorganism is deleted. Meanwhile, when a lactic acid-based copolymer comprising lactic acid and hydroxyalkanoic acid other than lactic acid as constitutional units is synthesized, hydroxyalkanoic acid other than lactic acid may be added to the medium, or the biosynthetic pathway for hydroxyalkanoic acid other than lactic acid may be provided for the host microorganism.
In particular, the recombinant microorganism according to the present invention produces aliphatic polyester oligomers outside the cells without accumulating aliphatic polyester within the cells. The recombinant microorganism of the present invention accumulates aliphatic polyester outside the cells, so that there is no need to increase cell growth efficiency in order to improve aliphatic polyester productivity. Therefore, the recombinant microorganism according to the present invention can produce aliphatic polyester oligomers at high levels even if a medium containing nutrient components to a degree such that growth is barely possible is used. Therefore, the recombinant microorganism according to the present invention is used so that high aliphatic polyester oligomer productivity can be achieved at low cost.
On the other hand, in the case of a recombinant microorganism that accumulates aliphatic polyester within cells, a policy employed herein to improve aliphatic polyester productivity involves increasing the growth efficiency of the recombinant microorganism and thus increasing the microbiomass. In this case, a medium with a high nutritional value should be used for increasing the growth efficiency of such a recombinant microorganism, resulting in very high cost. Also, in the case of a recombinant microorganism that accumulates aliphatic polyester within cells, culture must be completed at relatively early phase of the accumulation of aliphatic polyester within cells.
In contrast, the recombinant microorganism according to the present invention produces aliphatic polyester oligomers outside the cells, so that culture can be continued over a long time period and aliphatic polyester oligomers can be produced. Particularly in the case of the recombinant microorganism according to the present invention, fed-batch culture is preferably performed, comprising removing a portion from the medium and adding additional medium or some of medium components while continuing culture.
Meanwhile, when the recombinant microorganism according to the present invention is cultured for production of aliphatic polyester oligomers, low-cost medium containing general carbon sources and the like, such as minimal medium, is preferably used, but examples are not particularly limited thereto. Examples of carbon sources include carbohydrates such as glucose, fructose, sucrose, and maltose. Also, substances associated with fats and oils having a carbon number of 4 or more can also be used as carbon sources. Examples of a substance associated with fats and oils having a carbon number of 4 or more include natural fats and oils such as corn oil, soybean oil, safflower oil, sunflower oil, olive oil, coconut oil, palm oil, rape-seed oil, fish oil, whale oil, pig oil, and beef tallow oil, fatty acids such as butanoic acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, and myristic acid, or esters thereof, and alcohols such as octanol, lauryl alcohol, oleyl alcohol, and palmityl alcohol, or esters thereof.
Examples of nitrogen sources include, in addition to ammonia and ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and corn steep liquor. Examples of an inorganic material include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
Culture is preferably performed under aerobic conditions such as general shaking culture within a temperature range of 25° C.-37° C. for preferably 48 hours or more after the expression of the above pct gene and PHA synthase gene. During culture, antibiotics such as kanamycin, ampicillin, or tetracycline may be added to the medium. When either one of or both of the above pct gene and PHA synthase gene are introduced under control of an inducible promoter, a factor for inducing transcription from the promoter is added to the medium, and then culture is preferably performed for at least 72 hours.
In particular, lactic acid oligomers are preferably produced by culturing recombinant Escherichia coli in which the above pct gene and PHA synthase gene have been introduced. This method is advantageous in production cost, since lactic acid oligomers can be produced without adding a monomer component (e.g., lactic acid) composing a target polymer to the medium.
In addition, an aliphatic polyester oligomer such as a lactic acid oligomer can be recovered by a method known by persons skilled in the art. For example, cells are collected from a culture solution by centrifugation so as to remove cell components, and thus an aliphatic polyester oligomer such as a lactic acid oligomer can be recovered from the medium after removal of the cells according to a conventional method. The thus recovered product can be confirmed to be an aliphatic polyester oligomer such as a lactic acid oligomer by a general method such as gas chromatography or a nuclear magnetic resonance method.
Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited thereto.
In this Example, lactic acid oligomer productivity was evaluated for various PHA synthase genes when the genes had been expressed with a Megasphaera elsdenii-derived pct gene.
First, a pTV118N-M.E PCT vector for introduction of the Megasphaera elsdenii-derived pct gene was constructed. The M. elsdenii (ATCC17753) genome was obtained by a conventional method, and then the pct gene was obtained by a PCR method. As primers for amplification of a DNA fragment containing the M. elsdenii-derived pct gene, MePCTN: 5′-atgagaaaagtagaaatcattac-3′(SEQ ID NO: 19) and MePCTC:5′-ttattttttcagtcccatgggaccgtcctg-3′(SEQ ID NO: 20) were used. In addition, the nucleotide sequences of the primers were prepared with reference to the sequences disclosed in WO02/42418.
The pct gene was amplified from the genome under the following PCR conditions (enzyme KOD plus) (94° C. for 1 min)×1, (94° C. for 0.5 min, 50° C. for 0.5 min, 72° C. for 2 min)×30, and (94° C. for 2 min). The amplification fragment was introduced into a TOPO BluntII vector, and then sequencing was performed. As a result, the reported sequence had 97.8% homology with the nucleotide sequence, and only one portion thereof differed from the amino acid sequence.
The M. elsdenii-derived pct gene obtained as described above by PCR was inserted between EcoR 1 and Pst I of a pTV118N vector (Takara Bio Inc.), so that a pTV118N-M.E PCT expression plasmid was constructed.
Next, the PHA synthase genes examined in this Example are listed in Table 2. In Table 2, regarding No. 1 (Rhodobacter sphaeroides) and No. 4 (Rhodospirillum rubrum), a plurality of genes registered under different accession numbers have been discovered, so that a plurality of genes were examined.
Rhodobacter
sphaeroides
Azorhizobium
caulinodans
Rhizobium etli
Rhodospirillum
rubrum
Colwellia
psychrerythraea 34H
Chromobacterium
violaceum
Pseudomonas sp. 61-3
Hyphomonas
neptunium
Haloquadratum
walsbyi
Haloarcula
marismortui
Synechocystis sp.
Alcanivorax
borkumensis SK2
Bacillus cereus
Acinetobacter
baumannii ATCC
Magnetospirillum
magneticum AMB-1
Xanthomonas
campestris pv.
Campestris
Ralstonia eutropha
In addition, in Table 2, Class I means that the PHA synthase gene has strong activity and has high substrate specificity, Class II means that the PHA synthase gene has low substrate specificity, and has weak activity, Class III means that the PHA synthase gene further requires the presence of phaE for PHA synthase reaction, and Class IV means that the PHA synthase gene further requires the presence of phaR for PHA synthase reaction.
DNA fragments containing 19 types of PHA synthase gene derived from 17 types of microorganism (shown in No. 1 to No. 17) were amplified by 1 cycle of PCR or 2 cycles of PCR. The DNA fragments were introduced into pTV188N vectors in which the Megasphaera elsdenii-derived pct gene had been introduced. Primers for 1st PCR designed for amplification of the DNA fragments are shown in Table 3 and Table 4.
Rhodobacter
R. sphae-YP
RsphaeroidesF
sphaeroides
RsphaeroidesR
R. sphae-ABA
Rhodobacter-fwd 2
Rhodobacter-rvs 2
Azorhizobium
A. cauli
Azorhizobium-fwd 3
aulinodans
Azorhizobium-rvs 3
Rhizobium
R. etil
Rhizobium-fwd 2
etli CFN 42
Rhizobium-rvs 2
Rhodospirillum
R. rubru-AAD
R. rubrumF
rubrum
R. rubrumR
R. rubru-CAB
Colwellia
C. psych
ColwelliaF
psychrerythraea
ColwelliaR
Chromobacterium
C. viola
C. violaceumF
violaceum
C. violaceumR
Pseudomonas
Ps61-3.C2
P. sp. 61-3 (phaC2)-fwd 2
P. sp. 61-3 (phaC2)-rvs 2
Hyphomonas
H. neptu
neptunium
Haloquadratum
H. walsb
HwalsbphaEC1stFwd
walsbyi
HwalsbphaEC1stRvs
Haloarcula
H. maris
HmarisphaEC1stFwd
marismortui
HmarisphaEC1stRvs
HaloarculaPhaEF
HaloarculaPhaER
Synechocystis
S. sp.
SynecphaEC1stFwd
SynecphaEC1stRvs
Alcanivorax
A. borku-YP
A. borkumensisF
borkumensis SK2
A. borkumensisR
Bacillus cereus
B. cereus
BcereusphaC1stFwd
BcereusphaC1stRvs
BcereusphaR1stFwd
BcereusphaR1stRvs
Acinetobacter
A. bauma
A. baumanniiF
baumannii
A. baumanniiR
Magnetospirillum
M. magne-BAE
M. magneticumF
magneticum AMB-1
M. magneticumR
Xanthomonas
X. campe
X. campestrisF
campestris
X. campestrisR
Ralstonia
R. eutro
eutropha H16
Primers for 2nd PCR designed for amplification of the DNA fragments are shown in Table 5 and Table 6.
Rhodobacter
R. sphae-YP
RYP3543372ndFwd
sphaeroides
RYP3543372ndRev
R. sphae-ABA
RABA795572ndFwd
RABA795572ndRev
Azorhizobium
A. cauli
Azorhizo-fwd
caulinodans
Azorhizo-rvs
Rhizobium etli
R. etil
Rhizo-fwd
Rhizo-rvs
Rhodospirillum
R. rubru-AAD
RrubruAAD2ndFwd
rubrum
RrubruAAD2ndRvs
R. rubru-CAB
Rhodospirillum-fwd
Rhodospirillum-rvs
Cotwellia
C. psych
Colwellia2ndFwd
psychretythraea
Colwellia2ndRev
Chromobacterium
C. viola
CviolaphaC2ndFwd
violaceum
CviolaphaC2ndRvs
Pseudomonas
Ps61-3.C2
PspC22ndFwd
PspC22ndRvs
Hyphomonas
H. neptu
Hypho-fwd
neptunium
Hypho-rvs
Haloquadratum
H. walsb
HwalsbphaEC2ndFwd
walsbyi
HwalsbphaEC2ndRvs
Haloarcula
H. maris
HmarisphaEC2ndFwd
marismortui
HmarisphaEC2ndRvs
Synechocystis
S. sp.
SynecphaEC2ndFwd
SynecphaEC2ndRvs
Alcanivorax
A. borku-YP
Aborku2ndFwd
borkumensis SK2
Aborku2ndRvs
Bacillus cereus
B. cereus
BcereusphaRC2ndFwd
BcereusphaRC2ndRvs
Acinetobacter
A. bauma
Abauma2ndFwd
baumannii
Abauma2ndRvs
Magnetospirillum
M. magne-BAE
Mmagne2ndFwd
magneticum AMB-1
Mmagne2ndRvs
Xanthomonas
X. campe
Xanthomonas2ndFwd
campestris
Xanthomonas2ndRev
Ralstonia
R. eutro
Reutro2ndfwd
eutropha H16
Reutro2ndrvs
Also, conditions for PCR using these primers are shown in Table 7 and Table 8.
In addition, the compositions A to H of reaction solutions under the reaction conditions shown in Table 7 and Table 8 are shown in Table 9.
In addition, regarding No. 13 (pha gene), 2 genes (phaR and phaC) were present sandwiching other genes. Hence, the genes were separately cloned by 1st PCR and then the resultants were linked to form a sequence by 2nd PCR. Furthermore, for ligation to a vector, PCR was performed again (composition of reaction solution: G′; temperature conditions: 94° C. for 2 minutes→94° C. for 15 seconds, 50° C. for 30 seconds, 68° C. for 1 minute and 40 seconds×5 cycles→94° C. for 15 seconds, 60° C. for 30 seconds, 68° C. for 1 minute and 40 seconds×30 cycles→68° C. for 5 minutes).
Also, for Nos. 2, 3, and 8 (phaC genes), each of the purified 2″ PCR products and a pTV118N-PCT-C1 vector were digested with restriction enzymes (Xba I and Pst I (Takara Bio Inc.)) and then loaded on agarose gel (0.8%, TAE) together with 10× loading buffer (Takara Bio Inc.), followed by separation by electrophoresis, excision, and purification. Purification was performed using a MinElute Gel Extraction Kit (QIAGEN) according to protocols. Ligation and transformation were each performed according to protocols using Ligation-Convenience Kit (Nippon Gene Co., Ltd.) and ECOS competent E. coli JM109 (Nippon Gene Co., Ltd.). The thus obtained transformant was cultured in 2 ml of LB-Amp medium, and then plasmid extraction was performed using a QIAprep Spin Miniprep Kit (QIAGEN). Sequence reaction was performed using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and then the sequences were confirmed using a DNA sequencer 3100 Genetic Analyzer (Applied Biosystems).
Furthermore, for Nos. 1, 4-7, and 9-17 (phaC genes), ligation was performed using an In-Fusion 2.0 Dry-Down PCR Cloning Kit (Clontech Laboratories) in view of simpleness for experimental protocols or the presence of a Pst I site within each phaC gene (Nos. 4, 6, 10, and 12). The other portions were subjected to procedures similar to the above.
Various phaC genes obtained above were each incorporated into pTV118N-M.E PCT, so that a vector was obtained. The thus obtained vector was introduced into Escherichia coli W3110 competent cells, so that recombinant Escherichia coli expressing a Megasphaera elsdenii-derived pct gene and any one of the above PHA synthase genes was prepared. The thus obtained recombinant Escherichia coli was plated on LB medium containing ampicillin, followed by static culture overnight at 37° C. The thus obtained colonies were plated on 2 mL of LB liquid medium containing ampicillin, and then shake culture was performed within a test tube at 37° C. until OD600 reached 0.6 to 1.0. Thus, the resultant was used as a pre-culture solution.
Next, the pre-culture solution (2 mL) was added to 200 mL of M9 medium containing ampicillin, 2% glucose, and 0.1 mM IPTG, and then rotation culture was performed using a 500-mL buffled Erlenmeyer flask at 30° C. for 48 hours at 130 rpm.
After completion of culture, the culture solution was transferred to a 50-mL corning tube, cells were collected under conditions of 3000 rpm and 15 minutes, and thus a supernatant was obtained. The culture solution (200 μl) was transferred to a pressure-proof reaction tube, and then 1.6 mL of chloroform was added. Furthermore, 1.6 mL of a mixed solution of methanol and sulfuric acid (methanol:sulfuric acid=17:3 (volume ratio)) was added, followed by 3 hours of refluxing within a water bath set at 95° C. Subsequently, the pressure-proof reaction tube was removed and then cooled to room temperature. The solution within the tube was then transferred to a test tube. Ultrapure water (0.8 mL) was further added to the test tube, the solution was mixed using a vortex, and then left to stand. After the solution was sufficiently left to stand, the chloroform phase of the lower layer was fractionated using a Pasteur pipette. The chloroform phase was filtered with a 0.2-μm mesh organic solvent-resistant filter, the resultant was transferred to a vial bottle for GC-MS, and thus a sample for analysis was obtained.
As a GC-MS apparatus, HP6890/5973 (Hewlett-Packard Company) was used. As a column, BD-1 122-1063 (inner diameter: 0.25 mm; length: 60 m; membrane thickness: 1 μm (Agilent Technology)) was used. Temperature increase conditions employed herein comprise maintaining the temperature at 120° C. for 5 minutes, increasing the temperature at 10° C./min to 200° C., increasing the temperature at 20° C./min to 300° C., and then maintaining the temperature for 8 minutes.
Meanwhile, Table 10 shows the results of examining lactic acid oligomer productivity using a kit for component determination by an enzyme method, F-Kit series (Roche Diagnostics).
As shown in Table 10, it was revealed that recombinant Escherichia coli in which the Alcanivorax borkumensis-derived PHA synthase gene (No. 12), the Hyphomonas neptunium-derived PHA synthase gene (No. 8), the Rhodobacter sphaeroides-derived PHA synthase gene (No. 1), the Rhizobium etli-derived PHA synthase gene (No. 3), the Pseudomonas sp.-derived PHA synthase gene (No. 7), or the Haloarcula marismortui-derived PHA synthase gene (No. 10) had been introduced had good lactic acid oligomer productivity.
Based on the results shown in
As an ESI-MS system, Q-TOF (Micromass) was used. The ionization method was electrospray ionization, and the ionization mode was negative ion mode. The capillary voltage was 3200 V, the cone voltage was 30 V, the ion source temperature was 80° C., and the desolvation temperature was 120° C. The method used for introducing a sample was an infusion method (direct introduction). Each sample was introduced at 5 μl/min. Also, the number of instances of integration (integration frequency) was 100 times.
The results of measuring lactic acid dimer, trimer, tetramer, and pentamer levels in medium for recombinant Escherichia coli in which any one of the Hyphomonas neptunium-derived PHA synthase gene (No. 8), the Rhodobacter sphaeroides-derived PHA synthase gene (No. 1), the Rhizobium etli-derived PHA synthase gene (No. 3), the Pseudomonas sp.-derived PHA synthase gene (No. 7), and the Haloarcula marismortui-derived PHA synthase gene (No. 10) had been introduced are shown in
Furthermore, the results of measuring lactic acid dimer, trimer, tetramer, pentamer, hexamer, and heptamer levels in medium for recombinant Escherichia coli in which the Alcanivorax borkumensis-derived PHA synthase gene (No. 12) had been introduced are shown in
In this Example, differences in lactic acid oligomer productivity depending on medium type were examined using recombinant Escherichia coli prepared in Example 1 through introduction of the Alcanivorax borkumensis-derived PHA synthase gene (No. 12).
In this Example, a lactic acid oligomer was produced in medium in a manner similar to that in Example 1 except for using M9 medium (hereinafter, M9YE medium) prepared as medium with a high nutritional value by adding an yeast extract and M9 medium as medium with a low nutritional value. The lactic acid oligomer quantity was determined by GC-MS. In addition, M9 medium contained 6.8 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, and 1 g of NH4Cl per liter thereof, and further contained 2 ml of 1M MgSO4, 100 ml of 20% glucose, 1 ml of 1% thiamine, and 0.1 ml of 1M CaCl2.
A yeast extract (1 g) was added to 1 l of each M9YE medium.
In this Example, the relationship between the time for culture and lactic acid oligomer productivity was examined using recombinant Escherichia coli prepared in Example 1 through introduction of the Alcanivorax borkumensis-derived PHA synthase gene (No. 12).
In this Example, a lactic acid oligomer was produced in medium in a manner similar to that in Example 1 except for continuing culture for 192 hours, and then the lactic acid oligomer quantity was determined by GC-MS.
It was similarly concluded on the basis of the results of this Example that the other recombinant Escherichia coli cells prepared in Example 1 maintain lactic acid oligomer productivity at high levels over long periods of time, for example. Therefore, it was revealed that a lactic acid oligomer can be produced at low cost through the use of recombinant Escherichia coli prepared in Example 1.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-069688 | Mar 2010 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/057077 | 3/24/2011 | WO | 00 | 11/1/2012 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2011/118671 | 9/29/2011 | WO | A |
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| 20110008855 | Park et al. | Jan 2011 | A1 |
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| 2471910 | Jul 2012 | EP |
| 3536050 | Mar 2004 | JP |
| 2005-295993 | Oct 2005 | JP |
| 2009-504146 | Feb 2009 | JP |
| 2011-024503 | Feb 2011 | JP |
| 2009-0026035 | Mar 2009 | KP |
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| 20130045516 A1 | Feb 2013 | US |
