WO2016084857A1 - Method for producing 3-hydroxy propionic acid, and transformant - Google Patents

Abstract

　Provided are: a method for producing 3-HP at high productivity without requiring neutralization by an alkali, using an S. pombe transformant; and an S. pombe transformant suitable for use in said method.　This method for producing 3-hydroxy propionic acid is characterized by using a Schizosaccharomyces pombe as a host, culturing a transformant having an exogenous malonyl CoA reductase gene and an exogenous acetyl CoA carboxylase gene in a liquid culture medium containing acetic acid or acetate and having a total concentration thereof of 10-50mM, to obtain 3-hydroxy propionic acid from said liquid culture medium.

Description

Method for producing 3-hydroxypropionic acid, and transformant

The present invention uses 3-hydroxypropionic acid (3) by using a transformant incorporating a foreign gene using Schizosaccharomyces pombe (hereinafter sometimes referred to as “S. pombe”) as a host. -To a method for producing HP).

3-HP is a very useful compound as a raw material for various chemical products such as acrylic esters. 3-HP can be produced through a fermentation process with microorganisms. For example, in a microorganism into which a gene encoding malonyl CoA reductase (hereinafter also referred to as MCR) (hereinafter also referred to as MCR gene) is introduced, 3-HP is produced from malonyl CoA. In addition, in a microorganism into which a gene encoding glycerol dehydratase, aldehyde dehydrogenase or the like has been introduced, 3-HP is produced from glycerin via 3-hydroxypropionaldehyde (3-HPA).

When 3-HP is produced by a microorganism, the culture medium becomes acidic due to the produced 3-HP. When the acid resistance of the microorganism is low, the growth of the microorganism itself is suppressed as the pH of the culture medium decreases, and as a result, the production amount of 3-HP also decreases. It is necessary to culture while neutralizing. However, when neutralized, since 3-HP is recovered as a salt, a step for converting to 3-HP is required. Thus, since 3-HP production is possible without the need for neutralization, microorganisms that produce 3-HP are preferably those with high acid resistance. For example, Patent Document 1 discloses S.A. A transformant having a gene encoding a foreign MCR gene and a foreign acetyl-CoA carboxylase (hereinafter also referred to as ACC) (hereinafter also referred to as ACC gene) introduced into pombe is cultured without neutralization. Thus, it was described that 3-HP was produced.

On the other hand, in a heterologous protein production system using eukaryotic microorganisms such as yeast, in order to improve the production efficiency of the desired heterologous protein, part or all of the genome part of the host that is unnecessary or harmful to heterologous protein production is removed. It is known to use improved hosts that have been deleted or inactivated. For example, Patent Document 2 discloses S.I. In Pombe, it is described that the production efficiency of heterologous proteins can be improved by using an improved host in which at least one gene selected from genes encoding a specific protease is deleted or inactivated.

International Publication No. 2013/137277 International Publication No. 2007/015470

In the present invention, S.M. A method for producing 3-HP with high productivity without the need for neutralization with alkali using a Pombe transformant, and S. cerevisiae suitable for the method. An object is to provide a transformant of pombe.

The method for producing 3-HP according to the present invention is described in A transformant having an exogenous MCR gene and an exogenous ACC gene using Pombe as a host is cultured in a liquid medium containing acetic acid or acetate and the total concentration thereof being 10 to 50 mM. It is characterized in that 3-hydroxypropionic acid is obtained from the medium.
In the method for producing 3-HP according to the present invention, the transformant further has a gene (hereinafter also referred to as ACS gene) encoding a foreign acetyl-CoA synthetase (hereinafter also referred to as ACS). It is preferable.
In the method for producing 3-HP according to the present invention, the transformant is preferably a transformant from which at least one gene encoding a protease inherent in the host has been deleted or inactivated. . The gene encoding the deleted or inactivated protease is selected from the group consisting of a gene encoding a metalloprotease, a gene encoding a serine protease, a gene encoding a cysteine protease, and a gene encoding an aspartic protease. More preferred is a gene, and more preferred is a gene selected from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene.
In the method for producing 3-HP according to the present invention, the foreign gene is preferably incorporated into a chromosome, and the foreign gene is preferably incorporated into a plasmid.
In the method for producing 3-HP according to the present invention, the liquid medium preferably contains glucose or sucrose, and the total concentration thereof is 1 to 50% by mass.
In the method for producing 3-HP according to the present invention, it is preferable that the culture is further continued after the pH of the liquid medium is lowered to 3.5 or less due to the culture. It is also preferable to continue the culture without neutralizing the hydroxypropionic acid.

The transformant according to the present invention comprises Pombe is used as a host, it has a foreign MCR gene and a foreign ACC gene, and at least one gene encoding a protease inherent in the host is deleted or inactivated.
The transformant according to the present invention preferably further has a foreign ACS gene.
The transformant according to the present invention includes a gene encoding the deleted or inactivated protease, a gene encoding a metalloprotease, a gene encoding a serine protease, a gene encoding a cysteine protease, and an aspartic protease Preferably, it is a gene selected from the group consisting of genes encoding ps3, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene. Is more preferable.
In the transformant according to the present invention, the foreign gene is preferably integrated into a chromosome or a plasmid.

By the method for producing 3-HP according to the present invention, 3-HP can be produced efficiently by culturing the transformant without requiring neutralization treatment.
In addition, 3-HP can be efficiently produced by culturing the transformant according to the present invention.

1 is a schematic diagram of the structure of a pREP1-CaMCR-FLAG vector. 1 is a schematic diagram of the structure of a pREP2-cut6-FLAG vector. FIG. In Reference Example 1, it is a figure showing the results of an immunoblot using an anti-FLAG antibody of a transformant introduced with the pREP1-CaMCR-FLAG vector. In Reference Example 1, it is a figure showing the results of an immunoblot using an anti-FLAG antibody of a transformant introduced with the pREP2-cut6-FLAG vector. 1 is a schematic diagram of the structure of a 1-hsp9p-CaMCR-LPIT vector. FIG. 2 is a schematic diagram of the structure of a 2-hsp9p-cut6-LPIT vector. FIG. FIG. 3 is a schematic diagram of the structure of the pREP1-CaMCR vector. FIG. 4 is a schematic diagram of the structure of the pREP2-cut6 vector. FIG. 4 is a schematic diagram of the structure of the pREPAde-ACS2 vector. 1 is a schematic diagram of the structure of a pREPAde-acs1 vector. FIG.

The manufacturing method of 3-HP according to the present invention is S.I. A transformant having a foreign MCR gene and a foreign ACC gene using Pombe as a host is cultured in a liquid medium containing acetic acid or acetate, and 3-hydroxypropionic acid is obtained from the liquid medium. It is characterized by that. In the transformant having the MCR gene and the ACC gene, malonyl CoA is produced from acetyl CoA by ACC, and 3-HP is produced from this malonyl CoA by MCR. In addition, by culturing the transformant in the presence of acetic acid, the amount of acetyl CoA produced by acetic acid incorporated into the transformant increases, resulting in an increase in the amount of 3-HP produced. However, when the amount of acetic acid is too large, the production of 3-HP is suppressed. In the method for producing 3-HP according to the present invention, 3-HP can be produced efficiently by culturing the transformant in a liquid medium having a total concentration of acetic acid or acetate of 10 to 50 mM. .

[Transformant]
The transformant cultured in the method for producing 3-HP according to the present invention is S. cerevisiae. It is a transformant having a foreign MCR gene and a foreign ACC gene using Pombe as a host. By these foreign genes, 3-HP is produced from acetyl CoA in the transformant.

In the present invention and the present specification, a foreign gene is not a structural gene originally possessed by a host (a structural gene contained in a chromosome of a natural host before transformation), but a transformation operation, etc. Means the structural gene introduced by A gene introduced by a transformation operation or the like is a foreign gene in the present invention even if it is a gene originally possessed by the host.
Further, the foreign gene may be introduced into a chromosome, or may be incorporated into a plasmid and introduced into the cytoplasm.

<S. Pombe>
S. host. Pombe is a yeast belonging to the genus Schizosaccharomyces (fission yeast), and is a microorganism particularly excellent in acid resistance compared to other yeasts. S. Pombe is superior to other yeasts such as Saccharomyces cerevisiae to produce 3-HP under a high concentration of glucose, and is also suitable for high-density culture (culture using a large amount of yeast). I understood it. Therefore, S. By using a Pombe transformant, 3-HP can be produced with extremely high productivity.

S. The entire base sequence of the Pombe chromosome is recorded and published as “Schizosaccharomyces pombe Gene DB” (http://www.genedb.org/genedb/pombe/) in the database “GeneDB” of the Sanger Institute. As described in S.A. The sequence data of the pombe gene can be obtained by searching from the database by the gene name or the strain name.

<MCR gene>
The transformant used in the present invention has the MCR gene. S. Pombe does not naturally have the MCR gene. Therefore, S. The MCR gene derived from organisms other than Pombe can be obtained by genetic engineering. A transformant is obtained by introducing into pombe. Examples of the MCR gene introduced into the transformant include S. cerevisiae. It may be a structural gene that can express a protein that exhibits MCR activity when introduced into pombe, and may be an MCR gene derived from any species. Moreover, the foreign MCR gene possessed by the transformant may be one type or two or more types.

Examples of the MCR encoded by the MCR gene introduced as a foreign gene include, for example, Chloroflexus aurantiacus, Chloroflexus 、 aggregans, Roseiflexus castenholzii, and Roseiflexus.・ SP (Roseiflexus sp.), Roseiflexus SP ・ Strain RS-1 (Roseiflexussp. Strain RS-1), Erythrobacter sp., Erythrobacter strain NAP1 (Erythrobactersp. Strain NAP1), Gamma・ Proteobacterium (gamma proteobacterium), Metallosphaera sedula, Sulfolobus tokodaii, Sulfolobus metallicus, Sulfolobus Suspolobus sp., Acidianus brierleyi, Acidianus infernos, Acidianus ambivalens, Stygiolobus robus, riculobususroro and MCR derived from fumarii). Moreover, the modified body of these MCR may be sufficient. Examples of the MCR variants include polypeptides having an MCR activity consisting of an amino acid sequence in which one or several amino acids of these MCR amino acid sequences are substituted, added, or deleted. A polypeptide comprising an amino acid sequence having an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more, and an MCR activity with these MCR amino acid sequences. It is done. The MCR possessed by the transformant used in the present invention is preferably MCR derived from Chloroflexus aurantiacus (CaMCR, SEQ ID NO: 1).

<ACC gene>
The transformant used in the present invention has a foreign ACC gene. Examples of the ACC gene introduced into the transformant include S. cerevisiae. It may be a structural gene that can express a protein that exhibits ACC activity when introduced into pombe, and may be an ACC gene derived from any species. Moreover, the number of the foreign ACC gene which this transformant has may be one, and two or more types may be sufficient as it.

Examples of ACC encoded by the ACC gene introduced as a foreign gene include S. Pombe, Saccharomyces cerevisiae, Bacillus cereus, Bacillus subtilis, Bos taurus, Brevibacterium thiogenitalis, Lactobacillus plantarum, plant Lactobacillus Candida lipolytica, Candida catenulata, Candida gropengiesseri, Candida rugosa, Candida sonorensis, Candida sonorensis (Candida andsonorensis) methanosorbosa, Candida curvata, Kluyveromyces marxianus, Kluyveromyces thermotolerans, I Ittchenkia orientalis, Rhodotorula sglutinis, Pichia haplophila, Aspergillus clavatus, Aspergillus フ ラ sperm, sperm Aspergillus terreus, Aspergillus oryzae, Aspergillus ochraceus, Aspergillus niger, Cryptosporidium parvros, gastrodia Homo sapiens, Escherichia coli, Escherichia blattae, hordeum Bardere (Hordeum vulgare), Mycobacterium avium, Mycobacterium phlei, Setaria viridis, Solanum tuberosum, Spinacia olerac (ea) Staphylococcus aureus, Streptomyces coelicolor, Sulfolobus metallicus, Takifugu esp., Propionibacterium shermanae, udomonas ), Pseudomonas fluorescens, Pseudomonas sp., Chloroflexus orientiacus, Chloroflexus agregan , Metallosfaela cedura, Rhodosporidium toruloides, Cryptococcus curvatus, Trichosporon cutaneum, Lipomyces starkeyi, and bacterium C. ACC derived from Moreover, the modified body of these ACC may be sufficient. Examples of the modified ACC include a polypeptide having an ACC activity, which is composed of an amino acid sequence in which one or several amino acids of these ACC amino acid sequences are substituted, added, or deleted. A polypeptide comprising an amino acid sequence having an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more, and an ACC activity with these ACC amino acid sequences. It is done. Examples of ACC possessed by the transformant used in the present invention include S. cerevisiae. Pombe-derived ACC (SEQ ID NO: 2) is preferred. S. The gene encoding Pombe ACC is the cut6 gene.

<ACS gene>
The transformant used in the present invention preferably has a foreign ACS gene in addition to the MCR gene and the ACC gene. When a sufficient amount of ACS is expressed in the transformant, the amount of acetyl CoA converted from acetic acid increases, resulting in an increase in the production of 3-HP.

Examples of ACS encoded by the ACS gene introduced as a foreign gene include S.I. Examples include ACS derived from Pombe and Saccharomyces cerevisiae. These ACS variants may also be used. ACS variants include, for example, polypeptides having an ACS activity consisting of an amino acid sequence in which one or several amino acids of these ACS amino acid sequences are substituted, added, or deleted. Polypeptides comprising an ACS amino acid sequence and an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more, and have ACS activity. It is done. The ACC possessed by the transformant used in the present invention includes Saccharomyces cerevisiae-derived ACS (ACS2, SEQ ID NO: 3) or S. cerevisiae. Pombe derived ACS (acs1, SEQ ID NO: 4) is preferred.

<Protease gene>
The transformant used in the present invention is preferably a transformant in which at least one of a gene encoding a protease (hereinafter also referred to as a protease gene) is deleted or inactivated. S. By inhibiting the protease activity of at least one protease gene inherent in Pombe, the production efficiency of MCR and ACC is improved, and the production amount of 3-HP is further increased. The transformant used in the present invention encodes a gene encoding serine protease (hereinafter also referred to as serine protease gene), a gene encoding aminopeptidase (hereinafter also referred to as aminopeptidase gene), and carboxypeptidase. A gene in which one or more genes selected from the group consisting of a gene (hereinafter also referred to as carboxypeptidase gene) and a gene encoding dipeptidase (hereinafter also referred to as dipeptidase gene) have been deleted or inactivated. preferable.

The transformant used in the present invention may be one in which only one type of protease gene has been deleted, or one in which two or more types of protease genes have been deleted. Among them, a transformant in which at least one gene selected from the group consisting of a metalloprotease gene, a serine protease gene, a cysteine protease gene, and an aspartic protease gene has been deleted is preferable. From the metalloprotease gene and the serine protease gene A transformant from which at least one gene selected from the group consisting of at least one gene selected from the group consisting of a cysteine protease gene and an aspartic protease gene has been deleted is also preferred.

S. Examples of the four protease genes of Pombe include the following. Metalloprotease genes: cdb4 (SPAC23H4.09), mas2 (SPBC18E5.12c), pgp1 (SPCC1259.10), ppp20 (SPAC4F10.02), ppp22 (SPBC14C8.03), ppp51 (SPAC22G7.01c), ppp52 (SPBC18A7. 01), ppp53 (SPAP14E8.04). Serine protease genes: isp6 (SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01), sxa2 (SPAC1296.03c). Cysteine protease genes: ppp80 (SPAC19B12.08), pca1 (SPCC1840.04), cut1 (SPCC5E4.04), gpi8 (SPCC11E10.02c). Aspartic protease genes: sxa1 (SPAC26A3.01), yps1 (SPCC1795. 09), ppp81 (SPAC25B8.17).

The metalloprotease gene deleted in the transformant used in the present invention is preferably at least one selected from the group consisting of cdb4 gene, pgp1 gene, ppp20 gene, ppp22 gene, ppp52 gene and ppp53 gene, and cdb4 More preferred is at least one selected from the group consisting of a gene, a ppp22 gene, and a ppp53 gene.
The serine protease gene deleted in the transformant used in the present invention is preferably at least one selected from the group consisting of the isp6 gene, the ppp16 gene, the psp3 gene and the sxa2 gene.
As the cysteine protease gene deleted in the transformant used in the present invention, the ppp80 gene is preferable.

The transformant used in the present invention is at least one gene selected from the group consisting of cdb4 gene, ppp22 gene and ppp53 gene, and at least selected from the group consisting of isp6 gene, ppp16 gene, psp3 gene and sxa2 gene. Those in which 3 or more genes in total of 2 types of genes are deleted are preferable, and a total of 3 or more types including at least one gene selected from the group consisting of ppp53 gene and cdb4 gene, isp6 gene and psp3 gene Those in which the gene is deleted are more preferred. For example, those in which at least three genes of psp3 gene, isp6 gene and ppp53 gene are deleted are more preferable.

The transformant used in the present invention is preferably one in which four or more genes including the ppp53 gene, isp6 gene, psp3 gene and ppp16 gene are deleted, and the ppp53 gene, isp6 gene, psp3 gene, More preferably, 5 or more types of genes including the ppp16 gene and the ppp22 gene are deleted, and 6 or more types of genes including the ppp53 gene, isp6 gene, psp3 gene, ppp16 gene, ppp22 gene and sxa2 gene are deleted. More preferably, the psp3 gene, the isp6 gene, the ppp53 gene, the ppp16 gene, the ppp22 gene, the sxa2 gene, the ppp80 gene and the ppp20 gene are deleted. Preferred.

<Manufacture of transformant>
The transformant according to the present invention is a host strain of S. cerevisiae. It can be produced by introducing an MCR gene and an ACC gene (if necessary, an ACS gene) into a pombe by a genetic engineering method. When a plurality of foreign genes are introduced, all of them may be introduced simultaneously into the host or sequentially (in any order). Regarding gene recombination methods using yeast as a host, various expression systems, particularly expression vectors, secretion signal gene-introduced expression vectors, and the like have been developed in order to more stably and efficiently express heterologous proteins. These can be widely applied in the production of such transformants. For example, S.M. Examples of expression systems using pombe as a host include Japanese Patent No. 2776085, Japanese Patent Application Laid-Open No. 07-163373, Japanese Patent Application Laid-Open No. 10-215867, Japanese Patent Application Laid-Open No. 10-215867, Japanese Patent Application Laid-Open No. -192094, JP-A-2000-262284, WO 96/023890, etc. are known, and these expression systems can be widely used in the method for producing the transformant according to the present invention.

S. host The pombe may be a wild type or a mutant type in which a specific gene is deleted or inactivated depending on the use. As a method for deleting or inactivating a specific gene, a known method can be used. Specifically, the gene can be deleted by using the Latour method (described in Nucleic Acids Res, 2006, 34, e11, International Publication No. 2007/063919). In addition, mutation isolation methods using mutants (Yeast Molecular Genetics Experimental Method, 1996, Society Press Center) and random mutation methods using PCR (PCR Methods Application, Vol. 2, pages 28-33, 1992) etc., the gene can be inactivated by introducing a mutation into a part of the gene. In addition, the part where a specific gene is deleted or inactivated may be an ORF (open reading frame) part or an expression regulatory sequence part. A particularly preferred method is a method of deletion or inactivation by PCR-mediated homologous recombination method (Yeast, Vol. 14, pages 943-951, 1998) in which the ORF portion of the structural gene is replaced with a marker gene.

Examples of yeast hosts of the genus Schizosaccharomyces from which a specific gene has been deleted or inactivated are described in, for example, International Publication No. 2002/101038, International Publication No. 2007/015470, International Publication No. 2013/137277, etc. . Examples of the host used in the present invention include S. cerevisiae. Preferably, at least one protease gene originally possessed by Pombe is deleted or inactivated, and at least one gene selected from the group consisting of a serine protease gene, an aminopeptidase gene, a carboxypeptidase gene, and a dipeptidase gene is present. More preferably deleted or inactivated, wherein at least one gene selected from the group consisting of metalloprotease gene, serine protease gene, cysteine protease gene and aspartic protease gene is deleted or inactivated Is more preferable, from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene Which at least one gene has been deleted or inactivated barrel more preferably more.

Furthermore, S. is used as a host. It is preferable to use a pombe having a marker for selecting a transformant. For example, it is preferable to use a host in which a specific nutritional component is essential for growth because a certain gene is missing. When a transformant is produced by transforming with a vector containing the target gene sequence, by incorporating this missing gene (an auxotrophic complementary marker) into the vector, the transformant is required for host nutrition. Sex disappears. Due to the difference in auxotrophy between the host and the transformant, a transformant can be obtained by distinguishing the two.

For example, a gene encoding orotidine phosphate decarboxylase (hereinafter also referred to as ura4 gene) has been deleted or inactivated and has become uracil-requiring. After transforming with a vector having the ura4 gene, which is an auxotrophic complementary marker, using pombe as a host, a transformant in which the vector is incorporated can be obtained by selecting those that have lost uracil requirement. The gene that becomes auxotrophic due to deletion in the host is not limited to the ura4 gene as long as it is used for selection of transformants, and a gene encoding isopropylmalate dehydrogenase (hereinafter also referred to as leu1 gene). It may be.

<Foreign gene introduction method>
Each foreign gene is S. cerevisiae. It is introduced into the pombe chromosome or plasmid. In particular, it is preferably introduced into the chromosome. By introducing a foreign gene into the chromosome, a transformant having excellent passage stability can be obtained. A plurality of foreign genes can also be introduced into the chromosome.

As a method of introducing a foreign gene into a host by a genetic engineering method, a known method can be used. The foreign gene is S. cerevisiae. As a method for introduction into the pombe chromosome, a method of introduction by homologous recombination using a vector having an expression cassette having a foreign gene and a recombination site is preferred.

<Expression cassette>
An expression cassette is a combination of DNAs necessary for expressing a target protein, and includes a structural gene encoding the target protein, a promoter functioning in the host, and a terminator. The expression cassette used in the production of the transformant used in the present invention is a foreign gene, S. cerevisiae or the like. A promoter that functions in Pombe Including a terminator that functions within the pombe. The expression cassette may contain any one or more of 5′-untranslated region and 3′-untranslated region. Furthermore, the auxotrophic complementary marker may be included. Multiple expression genes may be present in one expression cassette. The number of foreign genes in one expression cassette is preferably 1-8, and more preferably 1-5. When a plurality of foreign genes are included in one expression cassette, two or more types of foreign genes may be included. Specifically, in the production of the transformant, the MCR gene and the ACC gene may be introduced into the host by separate expression cassettes, or both genes may be introduced into the host by one expression cassette.

As the gene sequences of the MCR gene and the ACC gene (ACS gene if necessary) included in the expression cassette, the gene encoded by the wild type may be used as it is. In order to increase the expression level in pombe, the wild type gene sequence was transformed into S. cerevisiae. It may be modified to a codon frequently used in pombe.

S. Promoters and terminators that function in the pombe function in the transformant even if 3-HP accumulates by the transformant according to the present invention and become acidic (pH 6 or lower), and the foreign gene is encoded. Any protein can be used as long as it can maintain the expression of the protein. S. Examples of promoters that function in pombe include S. cerevisiae. Pombe's inherent promoter (preferably having high transcription initiation activity) or S. A promoter (eg, a virus-derived promoter) that Pombe does not originally have can be used. Two or more promoters may be present in the vector.

S. Examples of promoters inherent to Pombe include alcohol dehydrogenase gene promoter, nmt1 gene promoter involved in thiamine metabolism, fructose-1, 6-bisphosphatase gene promoter involved in glucose metabolism, and invertase gene involved in catabolite repression. Examples include promoters (see International Publication No. 99/23223 pamphlet), heat shock protein gene promoters (see International Publication No. 2007/26617 pamphlet, International Publication No. 2014/030644), and the like.
S. Examples of promoters that Pombe does not originally include are promoters derived from animal cell viruses described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375, and hCMV A promoter, SV40 promoter is preferred.

S. As a terminator functioning in the pombe, S. Pombe's inherent terminator and S.P. A terminator that Pombe does not have can be used. Two or more terminators may be present in the vector.
Examples of the terminator include human terminators described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375, and human lipocortin I terminator is preferable. .

<Vector>
The transformant used in the present invention has an expression cassette containing a foreign gene in the chromosome or as an extrachromosomal gene. Having an expression cassette in the chromosome means that the expression cassette is incorporated at one or more positions in the chromosome of the host cell, and having as an extrachromosomal gene means having a plasmid containing the expression cassette in the cell. That is. A transformant containing each expression cassette is used as a host using a vector containing each expression cassette. It is obtained by transforming pombe.

The vector can be produced by incorporating the expression cassette into a vector having a circular DNA structure or a linear DNA structure. When the expression cassette produces a transformant that is maintained as an extrachromosomal gene in the host cell, the vector may contain a sequence for replication in the host cell, that is, an autonomously replicating sequence (AutonomouslynomReplicating). Sequence: ARS) is preferred. On the other hand, when producing a transformant in which the expression cassette is integrated into the host cell chromosome, the vector is assumed to have a linear DNA structure and no ARS. It is preferable to be introduced into. For example, the vector may be a vector composed of linear DNA, or a vector having a circular DNA structure provided with a restriction enzyme recognition sequence for cleavage into linear DNA upon introduction into a host. When the vector is a plasmid having ARS, it can be introduced into the host after forming a linear DNA structure by deleting the ARS part or a linear DNA structure in which the function of ARS is inactivated by cleaving the ARS part.

The vector preferably has a marker for selecting a transformant. Examples of the marker include ura4 gene and leu1 gene.

The vector is S. cerevisiae. When introduced into the chromosome of Pombe, the recombination site of the vector is S. This is a site having a base sequence that allows homologous recombination to be performed on a target site for homologous recombination in the pombe chromosome. The target site is S. pneumoniae. This is a target site for integrating the expression cassette in the pombe chromosome. The target site can be freely set by setting the recombination site of the vector to a base sequence that allows homologous recombination to be performed on the target site.
The homology between the base sequence of the recombination site and the base sequence of the target site needs to be 70% or more. Further, the homology between the base sequence of the recombination site and the base sequence of the target site is preferably 90% or more, and more preferably 95% or more from the viewpoint that homologous recombination is likely to occur. By using a vector having the recombination site, the expression cassette is incorporated into the target site by homologous recombination.
The length (number of bases) of the recombination site is preferably 20 to 2000 bp. If the length of the recombination site is 20 bp or more, homologous recombination is likely to occur. Moreover, if the length of the recombination site is 2000 bp or less, it is easy to prevent the vector from becoming too long and causing homologous recombination to hardly occur. The length of the recombination site is more preferably 100 bp or more, and further preferably 200 bp or more. Further, the length of the recombination site is more preferably 800 bp or less, and further preferably 400 bp or less.

The vector may have other DNA regions in addition to the expression cassette and the recombination site. For example, a replication initiation region called “ori” necessary for replication in E. coli and an antibiotic resistance gene (neomycin resistance gene, etc.) can be mentioned. These are genes usually required when constructing a vector using Escherichia coli. However, the replication initiation region is preferably removed when the vector is integrated into the host chromosome as described later.

When integrating a foreign gene into a chromosome, the vector is S. cerevisiae. When introducing into a pombe cell, it is preferable to introduce it in a linear DNA structure. That is, in the case of a vector having a circular DNA structure such as a commonly used plasmid DNA, S. It is preferable to introduce into pombe cells.
In this case, the position for opening the vector having a circular DNA structure is within the recombination site. As a result, the recombination sites partially exist at both ends of the opened vector, and the entire vector is integrated into the target site of the chromosome by homologous recombination.
The vector may be constructed by a method other than the method of cutting a vector having a circular DNA structure, as long as it can have a linear DNA structure in which a part of the recombination site exists at each end.

As the vector, for example, plasmids derived from E. coli such as pBR322, pBR325, pUC118, pUC119, pUC18, and pUC19 are preferably used.
In this case, the plasmid vector used for homologous recombination preferably has a replication initiation region called “ori” that is necessary for replication in E. coli. Thereby, when integrating the vector mentioned above into a chromosome, the integration efficiency can be improved.
The method for constructing the vector from which the replication initiation region has been removed is not particularly limited, but the method described in JP-A-2000-262284 is preferably used. That is, a method is preferred in which a precursor vector in which a replication initiation region is inserted at the cleavage site in the recombination site is constructed so that the replication initiation region is excised at the same time as the linear DNA structure as described above. Thereby, a vector from which the replication initiation region has been easily removed can be obtained.
Further, by applying the expression vector and the construction method thereof described in JP-A-5-15380, JP-A-7-163373, WO96 / 23890, JP-A-10-234375, Alternatively, a method may be used in which a precursor vector having an expression cassette and a recombination site is constructed, and a vector used for homologous recombination is obtained by removing the replication initiation region from the precursor vector by a normal genetic engineering technique.

<Target site>
Target sites that incorporate the vector are S. cerevisiae. It may be present only at one location in the pombe chromosome, or may be present at two or more locations. When two or more target sites exist, S.P. The vector can be integrated at two or more positions on the pombe chromosome. When a plurality of foreign genes are included in one expression cassette, a plurality of foreign genes can be incorporated into one target site. Furthermore, an expression cassette can be incorporated into two or more target sites using two or more vectors having recombination sites corresponding to the respective target sites. In this manner, S. A plurality of foreign genes can be incorporated into the pombe chromosome, thereby increasing the expression level of MRC or ACC encoded by the foreign gene and improving 3-HP productivity. For example, an expression cassette containing an MRC gene is incorporated into a vector having a first target site, an expression cassette containing an ACC gene is incorporated into a vector having a second target site, The transformant according to the present invention can be obtained by transforming pombe as a host.

When incorporating an expression cassette into one target site, for example, the target site described in the method described in JP-A No. 2000-262284 can be used. Two or more vectors having different integration sites can be used to integrate the vectors into different target sites. However, this method is complicated when the vector is integrated at two or more sites on the chromosome.

If the base sequence parts that are substantially identical to each other in a plurality of locations in a chromosome can be used as target sites and the vectors can be incorporated into the target sites in these multiple locations, a vector can be used in two or more locations on the chromosome using one type of vector. Can be incorporated. Base sequences that are substantially identical to each other means that the homology of the base sequences is 90% or more. The homology between the target sites is preferably 95% or more. In addition, the length of the base sequences that are substantially identical to each other is a length that includes the recombination site of the vector, and is preferably 1000 bp or more. Compared to the case where multiple foreign genes are incorporated into one target site, even if the number of foreign genes incorporated is the same, the foreign genes are dispersed and incorporated into multiple target sites In this case, when the transformant grows, the foreign gene is less likely to drop off from the chromosome at a time, and the maintenance stability in the passage of the transformant is improved.

A transposon gene Tf2 is preferable as a target site present in a plurality of locations in a chromosome. Tf2 is the S.T. There are a total of 13 transposon genes in each of the three pombe (haploid) chromosomes, the length (number of bases) is about 4900 bp, and the base sequence homology between these genes is 99.7%. It is known (see the following literature).
Nathan J. Bowen et al, “Retrotransposons and Their Recognition of pol II Promoters: A Comprehensive Survey of the Transposable Elements From the Complete Genome Sequence of Schizosaccharomyces pombe”, Genome Res. 2003 13: 1984-1997

A vector can be incorporated into only one location of Tf2 present in 13 locations on a chromosome. In this case, a transformant having two or more foreign genes can be obtained by incorporating a vector having two or more foreign genes. In addition, a transformant having two or more foreign genes can be obtained by incorporating a vector into two or more locations of Tf2. In this case, a transformant having more foreign genes can be obtained by incorporating a vector having two or more foreign genes. If the vector is incorporated at all 13 positions of Tf2, the burden on the survival and growth of the transformant may be too great. Preferably, the vector is preferably incorporated at 8 sites or less of 13 Tf2, and more preferably at 5 sites or less.

<Transformation method>
Any known transformation method may be used as the transformation method. Examples of the transformation method include conventionally known methods such as lithium acetate method, electroporation method, spheroplast method, glass bead method, and the method described in JP-A-2005-198612. A commercially available yeast transformation kit may also be used.

In the production of a transformant, the obtained transformant is usually selected after homologous recombination. Examples of the selection method include the following methods. Screening is performed with a medium capable of selecting transformants using the auxotrophic marker, and a plurality of colonies obtained are selected. Next, after they are separately liquid cultured, the expression level of the heterologous protein in each liquid medium is examined, and a transformant with a higher expression level of the heterologous protein is selected. By performing genome analysis by pulse field gel electrophoresis on the selected transformants, the number of vectors integrated into the chromosome and the number of expression cassettes can be examined.
The number of vectors integrated into the chromosome can be adjusted to some extent by adjusting the integration conditions. Depending on the size (number of bases) and structure of the vector, the integration efficiency and the number of integrations may change.

[3-HP production]
In the method for producing 3-HP according to the present invention, the concentration of acetic acid or acetate in the liquid medium inoculating the transformant is higher than that in the case of culturing in a liquid medium to which acetic acid or the like is not added. The concentration may be any as long as the amount is high, and can be appropriately determined in consideration of the genotype of the transformant to be cultured and the other composition of the liquid medium, particularly the concentration of glucose or sucrose. The concentration of acetic acid or acetate in the liquid medium is preferably 10 to 50 mM, more preferably 10 to 40 mM, and further preferably 20 to 30 mM.

As the liquid medium used for the production of 3-HP, a known yeast culture medium containing sugar is used so that the concentration of acetic acid or acetate is within a predetermined range. Examples of the liquid medium include S.I. Contains a nitrogen source, inorganic salts, etc. that can be utilized by Pombe; Those capable of efficiently cultivating the pombe are preferred. As the liquid medium, a natural medium or a synthetic medium may be used.

Examples of the sugar that is a carbon source include sugars such as glucose, fructose, sucrose, and maltose. Examples of the nitrogen source include inorganic acids such as ammonia, ammonium chloride, and ammonium acetate, or ammonium salts of inorganic acids, peptone, casamino acid, yeast extract, and the like. Examples of inorganic salts include magnesium phosphate, magnesium sulfate, sodium chloride and the like. Furthermore, a fermentation promoting factor such as proteolipid can be included.

In the method for producing 3-HP according to the present invention, it is preferable to use a liquid medium containing glucose or sucrose as sugar. The concentration of glucose or sucrose in the liquid medium (100% by mass) at the initial stage of culture is preferably 1% by mass or more, more preferably 1 to 50% by mass, and further preferably 2 to 16% by mass. It is preferable to continue the culture by adding glucose as necessary, because the glucose concentration is lowered by the culture. The glucose concentration at the end of the culture may be 1% by mass or less. In addition, when continuously culturing by circulating a liquid medium while separating 3-HP, it is preferable to maintain the glucose concentration. By making the glucose concentration 2% by mass or more, the productivity of 3-HP is further improved. In addition, the production efficiency of 3-HP is further improved by setting the glucose in the liquid medium to 16% by mass or less.

Moreover, in order to increase the productivity of 3-HP production, it is preferable to perform high-density culture. In high-density culture, the initial bacterial cell concentration of the transformant in the liquid medium is preferably 0.1 to 5 g / L in terms of dry cell weight. More preferably, the initial cell concentration of the transformant in the liquid medium is 0.2 to 2 g / L in terms of dry cell weight. High productivity can be achieved in a short time by increasing the initial cell concentration. In addition, if the initial bacterial cell concentration is too high, problems such as bacterial cell aggregation and a reduction in purification efficiency may occur.

Incidentally, cell concentration indicated in the Examples etc. described later, the absorbance of light (OD ₆₀₀₎ values of wavelength 600nm was measured by JASCO Corporation Ltd. visible ultraviolet spectrometer V550.

A known yeast culture method can be used for the culture, for example, shaking culture, stirring culture, or the like.
The culture temperature is preferably 23 to 37 ° C. Further, the culture time can be determined as appropriate.
Further, the culture may be batch culture or continuous culture. For example, after culturing in batch culture, the cells can be separated from the liquid medium to obtain a liquid medium containing 3-HP. In the continuous culture method, for example, a part of the liquid medium is extracted from the culture tank being cultured, and 3-HP is separated from the extracted liquid medium, and the culture supernatant is recovered, and glucose and A method of continuously culturing by repeatedly adding a new liquid medium or the like and returning it to the culture tank can be mentioned. By performing continuous culture, the productivity of 3-HP is further improved.

In the method for producing 3-HP according to the present invention, S.I. Since the Pombe transformant is used, 3-HP can be produced without neutralization even if the pH is lowered due to the accumulation of 3-HP (pH of about 2 to 4). Therefore, even after the pH of the liquid medium becomes 3.5 or less, 3-HP can be produced by continuous culture that continues the culture. The pH at the end of culture and the pH in continuous culture are preferably 3.5 or less, and particularly preferably 2.3 to 3.5. In order to increase the productivity of 3-HP, it is preferable to continue the cultivation after the pH of the liquid medium becomes 3.5 or lower. Since the transformant used in the present invention has excellent acid resistance, the culture can be continued without neutralizing 3-HP in the liquid medium produced by the transformant.

For obtaining 3-HP from the liquid medium, a known method is used. In particular, it is preferable to obtain 3-HP by separating the liquid medium and 3-HP without neutralizing 3-HP in the liquid medium. For example, by separating the bacterial cells from the liquid medium after completion of the culture by centrifugation, extracting with diethyl ether or ethyl acetate after adjusting the pH to 1 or less, a method of leaching after adsorbing to an ion exchange resin and washing, activated carbon And a method of removing impurities using, a method of distilling after reacting with an alcohol in the presence of an acid catalyst, and a method of separating using a separation membrane. In some cases, 3-HP can be obtained by neutralizing 3-HP in the liquid medium and then separating the liquid medium and the 3-HP salt. For example, 3-HP can be obtained by converting 3-HP in a liquid medium into a calcium salt or a lithium salt and crystallizing the neutralized salt.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description. In this example, “%” means “% by mass” unless otherwise specified.

[Reference Example 1]
Wild type S. cerevisiae Pombe ARC039 strain (genotype: h ^- leu1-32 ura4-C190T) Among pombe, the MGF433 strain in which the psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene have been deleted (genotype: h ^- leu1-32 ura4-D18 psp3 :: ura4 (FOA) isp6 :: ura4 (FOA) ppp53 :: ura4 (FOA) ppp16 :: ura4 (FOA) ppp22 :: ura4 (FOA) sxa2 :: ura4 (FOA) ppp80 :: ura4 (p20: p20: p4 ) (see Patent Document 2) the FOA treated MGF438 strain was uracil auxotrophy and ^{(genotype: h - leu1-32 ura4-D18 psp3} :: ura4 (FOA) isp6 :: ura4 (FOA) ppp 3 :: ura4 (FOA) ppp16 :: ura4 (FOA) ppp22 :: ura4 (FOA) sxa2 :: ura4 (FOA) ppp80 :: ura4 (FOA) ppp20 :: ura4 (FOA)), CaMCR-FLAGCa The expression cassette of FLAG tag added to the C terminus of) or the expression cassette of cut6-FLAG (the FLAG tag added to the C terminus of S. pombe-derived ACC (cut 6)) was compared for expression. .

First, a recombinant vector pREP1-CaMCR-FLAG (12471 bp, FIG. 1) carrying a CaMCR-FLAG expression cassette was prepared. The pREP1-CaMCR-FLAG vector was prepared as a DNA fragment consisting of the base sequence shown in SEQ ID NO: 5 by DNA synthesis.
Similarly, a recombinant vector pREP2-cut6-FLAG (15166 bp, FIG. 2) carrying the cut6-FLAG expression cassette was prepared. The pREP2-cut6-FLAG vector was prepared as a DNA fragment consisting of the base sequence shown in SEQ ID NO: 6 by RT-PCR using RNA extracted from the MGF438 strain as a template.

Subsequently, the ARC039 strain and the MGF438 strain were transformed with the pREP1-CaMCR-FLAG vector, respectively. The transformant was streaked on an MM-leu plate (a plate obtained by removing leucine from an MM (minimum medium) plate) and cultured at 30 ° C., and 5 colonies were selected from the obtained colonies, and each MM-leu medium ( A medium obtained by removing leucine from the MM medium was inoculated and cultured at 30 ° C.
In addition, ARC039 strain and MGF438 strain were transformed with pREP2-cut6-FLAG vector, respectively. The transformant was streaked on an MM-ura plate (a plate obtained by removing uracil from the MM plate) and cultured at 30 ° C., and one of the obtained colonies was selected, and each MM-ura medium (MM medium to uracil) was selected. And was cultured at 30 ° C. A sample buffer for SDS-PAGE was added to the cells recovered from the obtained culture and incubated at 99 ° C. for 3 minutes to prepare a sample for SDS-PAGE. The SDS-PAGE sample was applied to a polyacrylamide gel, and transferred to a PVDF membrane after SDS-PAGE. Immunoblotting was performed on the PVDF membrane to which the protein was transferred using an HRP-labeled anti-FLAG antibody (Anti-DDDDK-tag mAb-HRP-DirecT, manufactured by Institute of Pharmaceutical Biology).

FIG. 3 shows the result of immunoblotting of the transformant introduced with the pREP1-CaMCR-FLAG vector, and FIG. 4 shows the result of immunoblotting of the transformant introduced with the pREP2-cut6-FLAG vector. As a result, in the transformant using ARC039 strain as a host, both CaMCR-FLAG and cut6-FLAG were significantly degraded by protease, and a large number of degradation products were detected. However, in the transformant using MGF438 strain as a host, In addition, degradation of both CaMCR-FLAG and cut6-FLAG was suppressed.

[Example 1]
S. Among the pombe, the MSP0080 strain (genotype: h ^- leu1-32 ura4-D18) in which the psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene, and ppp20 gene have been deleted by the Latour method psp3-D13 isp6-D14 oma1-D10 ppp16-D20 fma2-D13 sxa2-D15 aap1-D17 ppp80-D11) and the gene encoding CaMCR In producing 3-HP using a transformant incorporating the pombe-derived cut6 gene, the effects of glucose concentration and acetic acid concentration in the liquid medium used for cultivation on 3-HP production were examined.

<Preparation of MCR / ACC-1 expression strain>
First, a recombinant vector 1-hsp9p-CaMCR-LPIt (11052 bp, FIG. 5, SEQ ID NO: 7) in which a gene encoding CaMCR is incorporated between the hsp9 promoter and the LPI terminator and between the hsp9 promoter and the LPI terminator S. A recombinant vector 2-hsp9p-cut6-LPIt (13749 bp, FIG. 6, SEQ ID NO: 8) in which the pombe-derived cut6 gene was incorporated was prepared by DNA synthesis.
Subsequently, the MSP0080 strain was transformed with the 1-hsp9p-CaMCR-LPIT vector and the 2-hsp9p-cut6-LPIT vector, and transformants into which both vectors were introduced were selected. The transformant was designated as an MCR / ACC-1 expression strain.

<Preparation of MCR / ACC-2 expression strain and MCR / ACC / ACS2 expression strain>
First, a recombinant vector pREP1-CaMCR (12431 bp, FIG. 7, SEQ ID NO: 9) in which a gene encoding CaMCR is incorporated between the nmt1 promoter and the nmt1 terminator, and a cut6 gene is incorporated between the nmt1 promoter and the nmt1 terminator. Recombinant vector pREP2-de6 (15114 bp, FIG. 8, SEQ ID NO: 10) and recombinant vector pREPAde-ACS2 in which a gene encoding ACS2 derived from Saccharomyces cerevisiae is incorporated between the nmt1 promoter and the nmt1 terminator (10970 bp, FIG. 9, SEQ ID NO: 11) and a recombinant vector in which a gene encoding acs1 derived from S. pombe is incorporated between the nmt1 promoter and the nmt1 terminator REPAde-acs1 (10907bp, 10, SEQ ID NO: 12) and was produced by DNA synthesis.
Subsequently, a strain in which the ade7 gene of the MSP0080 strain was disrupted to impart adenine requirement was transformed with the pREP1-CaMCR vector and the pREP2-cut6 vector, and transformants into which all of the two vectors had been introduced were selected. The transformant was designated as an MCR / ACC-2 expression strain.
Subsequently, the MCR / ACC-2 expression strain was transformed with the pREPAde-ACS2 vector, and a transformant into which the vector was introduced was selected. The transformant was designated as an MCR / ACC / ACS2 expression strain. Further, the MCR / ACC-2 expression strain was transformed with the pREPAde-acs1 vector, and a transformant into which the vector was introduced was selected. The transformant was designated as an MCR / ACC / acs1 expression strain.

<3-HP production>
MCR / ACC-1 expression strain and MCR / ACC / ACS2 expression strain were cultured using a liquid medium having various concentrations of glucose and acetic acid, and the amount of 3-HP produced was measured and compared.

Specifically, the MCR / ACC-1 expression strain was inoculated into an MM-leu-ura medium (a medium obtained by removing leucine and uracil from the MM medium), and the temperature was 30 ° C. and the shaking speed was 200 rpm. Time culture was performed.
Subsequently, the cells are separated from the liquid medium after the completion of the culture by centrifugation, and the cells are added to the MM-leu-ura medium having the glucose concentration and the acetic acid concentration shown in Table 1, and the initial cell concentration is OD ₆₀₀ = 0.05) and cultured for 144 hours. The 3-HP concentration (mM) of the culture solution after completion of the culture was measured under the following HPLC conditions. Column used: Inert Sustain C18 5 μm, 4.6 ID × 250 mm
(Manufactured by GL Sciences)
Eluent: 10 mM NH ₄ H ₂ PO ₄ (pH 2.6, prepared with phosphoric acid)
Injection volume: 10 μl
Flow rate: 0.5ml / min
Column temperature: 30 ° C
Detection: UV (wavelength 210nm)

Similarly, the MCR / ACC / ACS2 expression strain was inoculated into an MM-leu-ura-adeneine medium (a medium obtained by removing leucine, uracil and adenine from the MM medium) at a temperature of 30 ° C. and a shaking speed of 200 rpm. For 30 hours.
Subsequently, the cells are separated from the liquid medium after the completion of the culture by centrifugation, and the cells are added to the MM-leu-ura-adeneine medium having the glucose concentration and the acetic acid concentration shown in Table 2, with the initial cell concentration being OD. ₆₀₀ = 0.05) and fermented for 144 hours. The 3-HP concentration (mM) of the fermentation broth after completion of fermentation was measured by HPLC.

Tables 1 and 2 show the measurement results of 3-HP concentration (mM) of each fermentation broth. As a result, in both strains, 3-HP production was higher when the concentration of acetic acid in the liquid medium inoculating the cells was 20 mM than when 0 mM, but the concentration of acetic acid was higher. When too much, 3-HP production was inhibited. In addition, when the acetic acid concentration is 20 mM, the amount of 3-HP produced increases depending on the glucose concentration. In particular, in the MCR / ACC-1 expression strain, when the acetic acid concentration is 20 mM and the glucose concentration is 15%, 0.033 mM of 3-HP was produced. In the MCR / ACC / ACS2 expression strain, 3-HP of 26 mM or more was produced when the acetic acid concentration was 30 mM and the glucose concentration was 12%, and when the acetic acid concentration was 40 mM and the glucose concentration was 6%.

[Example 2]
The MSP0080 strain contains a gene encoding CaMCR and S. cerevisiae. A transformant into which the pombe-derived cut6 gene was introduced; a gene encoding CaMCR; The amount of 3-HP produced by the transformants into which the pombe-derived cut6 gene and the gene encoding ACS2 from Saccharomyces cerevisiae were introduced was compared.

Specifically, the MCR / ACC-2 expression strain produced in Example 1 was inoculated into an MM-leu-ura medium and cultured for 30 hours under conditions of a temperature of 30 ° C. and a shaking speed of 200 rpm.
Subsequently, the cells are separated from the liquid medium after the completion of the culture by centrifugation, and the cells are first introduced into an MM-leu-ura medium (glucose concentration: 9%, acetic acid concentration: 20 mM) containing glucose and acetic acid. Fermentation was carried out for 144 hours using a fermented liquid that was inoculated so that the bacterial cell concentration was (OD ₆₀₀ = 0.05). The 3-HP concentration (mM) of the fermentation broth after completion of fermentation was measured by HPLC.

Further, the MCR / ACC / ACS2 expression strain produced in Example 1 was inoculated into an MM-leu-ura-adeneine medium and cultured for 30 hours under conditions of a temperature of 30 ° C. and a shaking speed of 200 rpm.
Subsequently, the cells are separated from the liquid medium after the culture by centrifugation, and the cells are placed in an MM-leu-ura-adeneine medium (glucose concentration: 9%, acetic acid concentration: 20 mM) containing glucose and acetic acid. The fermented broth was inoculated so that the initial bacterial cell concentration was (OD _6OO = 0.05), and fermented for 144 hours. The 3-HP concentration (mM) of the fermentation broth after completion of fermentation was measured by HPLC.

As a result, the 3-CR production amount of the MCR / ACC-2 expression strain was 15.9 mM, the 3-HP production amount of the MCR / ACC / ACS2 expression strain was 27.9 mM, and the MCR / ACC-2 expression strain The 3-HP production was improved 1.75 times in the MCR / ACC / ACS2 expression strain. From these results, it was found that the amount of 3-HP produced when fermented in the presence of acetic acid can be increased by overexpressing ACS2.

In addition, the MCR / ACC / acs1 expression strain produced in Example 1 was inoculated into an MM-leu-ura-adeneine medium and cultured for 30 hours under conditions of a temperature of 30 ° C. and a shaking speed of 200 rpm.
Subsequently, the cells are separated from the liquid medium after the culture by centrifugation, and the cells are placed in an MM-leu-ura-adeneine medium (glucose concentration: 9%, acetic acid concentration: 20 mM) containing glucose and acetic acid. The fermented broth was inoculated so that the initial bacterial cell concentration was (OD _6OO = 0.05), and fermented for 144 hours. The 3-HP concentration (mM) of the fermentation broth after completion of fermentation was measured by HPLC.

As a result, the 3-CR production amount of the MCR / ACC-2 expression strain was 18.7 mM, the 3-HP production amount of the MCR / ACC / acs1 expression strain was 29.3 mM, and the MCR / ACC-2 expression strain The 3-HP production was improved 1.56 times in the MCR / ACC / acs1-expressing strain. From these results, it was found that the amount of 3-HP produced when fermenting in the presence of acetic acid can be increased by overexpressing acs1.
It should be noted that the entire content of the specification, claims, abstract and drawings of Japanese Patent Application No. 2014-238629 filed on November 26, 2014 is cited here as the disclosure of the specification of the present invention. Incorporated.

Claims (15)

1. A transformant having a gene encoding exogenous malonyl-CoA reductase and a gene encoding exogenous acetyl-CoA carboxylase as a host, and containing acetic acid or acetate, and the total concentration thereof is Shizosaccharomyces pombe. A method for producing 3-hydroxypropionic acid, comprising culturing in a liquid medium of 10 to 50 mM and obtaining 3-hydroxypropionic acid from the liquid medium.
2. The method for producing 3-hydroxypropionic acid according to claim 1, wherein the transformant further has a gene encoding a foreign acetyl-CoA synthetase.
3. The production of 3-hydroxypropionic acid according to claim 1 or 2, wherein the transformant is a transformant in which at least one gene encoding a protease inherent in the host has been deleted or inactivated. Method.
4. The gene encoding the deleted or inactivated protease is selected from the group consisting of a gene encoding a metalloprotease, a gene encoding a serine protease, a gene encoding a cysteine protease, and a gene encoding an aspartic protease. The method for producing 3-hydroxypropionic acid according to claim 3, which is a gene.
5. The gene encoding the deleted or inactivated protease is a gene selected from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene. Item 5. The method for producing 3-hydroxypropionic acid according to Item 3 or 4.
6. The method for producing 3-hydroxypropionic acid according to any one of claims 1 to 5, wherein the foreign gene is integrated into a chromosome.
7. The method for producing 3-hydroxypropionic acid according to any one of claims 1 to 5, wherein the foreign gene is incorporated into a plasmid.
8. The method for producing 3-hydroxypropionic acid according to any one of claims 1 to 7, wherein the liquid medium contains glucose or sucrose, and the total concentration thereof is 1 to 50 mass%.
9. The method for producing 3-hydroxypropionic acid according to any one of claims 1 to 8, wherein the culture is further continued even after the pH of the liquid medium is lowered to 3.5 or less due to the culture.
10. The method for producing 3-hydroxypropionic acid according to any one of claims 1 to 9, wherein the culture is continued without neutralizing 3-hydroxypropionic acid in the liquid medium.
11. Schizo Saccharomyces pombe as a host, having a gene encoding a foreign malonyl-CoA reductase and a gene encoding a foreign acetyl-CoA carboxylase, and at least one gene encoding a protease inherent in the host is deleted or A transformant characterized by being inactivated.
12. The transformant according to claim 11, wherein the transformant further has a gene encoding a foreign acetyl-CoA synthetase.
13. The gene encoding the deleted or inactivated protease is selected from the group consisting of a gene encoding a metalloprotease, a gene encoding a serine protease, a gene encoding a cysteine protease, and a gene encoding an aspartic protease. The transformant according to claim 11 or 12, which is a gene.
14. The gene encoding the deleted or inactivated protease is a gene selected from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene. Item 14. The transformant according to any one of Items 11 to 13.
15. The transformant according to any one of claims 11 to 14, wherein the foreign gene is incorporated into a chromosome or a plasmid.

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