JP2017121184A - Mutant rhizopus bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide mutant Rhizopus bacteria having high organic acid productivity.SOLUTION: The present invention provides mutant Rhizopus bacteria having a reduced activity of alcohol dehydrogenase.SELECTED DRAWING: None

Description

  The present invention relates to a mutant Rhizopus sp. And a method for producing an organic acid using the same.

  Organic acids such as lactic acid, malic acid, succinic acid, and fumaric acid are substances with high industrial value, such as those used in food production and used as raw materials for synthetic resins and biodegradable polymers.

  In recent years, production of biologically useful substances using microorganisms and enzymes has been carried out. However, in the industrial production of substances by microorganisms, improvement of productivity is one of the important issues. In order to improve productivity, conventionally, breeding of producing bacteria by genetic techniques such as mutation has been performed. Particularly recently, with the development of microbial genetics and biotechnology, the development of more efficient microbiological material production methods using genetic recombination techniques has attracted attention.

  For example, Patent Documents 1 to 3 disclose a method for producing lactic acid using a microorganism into which a promoter of Rhizopus, which is a filamentous fungus, is introduced. Non-Patent Document 1 reports a mutant Rhizopus genus that produces a large amount of fumaric acid and a small amount of byproduct ethanol production. However, since the genetic background of Rhizopus spp. Has not yet been fully studied, it is not easy to develop Rhizopus spp. Suitable for production of useful substances by genetic recombination technology.

US Pat. No. 6,268,189 International Publication No. 2001/73083 International Publication No. 2001/72967

Korean J Chem Eng, 2010, 27 (1): 183-186

  The present invention relates to a mutant Rhizopus sp. Having high organic acid productivity, and a method for producing an organic acid using the same.

  The present inventors examined improvement of organic acid productivity of Rhizopus sp. As a result, the present inventors have found that organic acid productivity is remarkably improved in Rhizopus spp. In which the activity of alcohol dehydrogenase is reduced, and have completed the present invention.

  Accordingly, the present invention provides mutant Rhizopus spp. In which the activity of alcohol dehydrogenase is reduced.

  The present invention also provides a method for producing mutant Rhizopus, which comprises reducing the activity of alcohol dehydrogenase in Rhizopus.

  The present invention also provides a method for improving the organic acid productivity of Rhizopus bacteria, which comprises reducing the activity of alcohol dehydrogenase in Rhizopus bacteria.

  The present invention also provides a method for producing an organic acid, comprising culturing the mutant genus Rhizopus.

  The present invention provides a mutant genus Rhizopus having high organic acid productivity. According to the genus Rhizopus of the present invention, efficient microbiological production of an organic acid is realized.

  As used herein, nucleotide and amino acid sequence identity is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) as 2, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win.

  In the present specification, “at least 80% identity” with respect to an amino acid sequence or nucleotide sequence means 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably. 97% or more, preferably 98% or more, and more preferably 99% or more identity. In the present specification, “at least 85% identity” with respect to an amino acid sequence or nucleotide sequence means 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and still more preferably. An identity of 98% or more, more preferably 99% or more. In the present specification, “at least 95% identity” with respect to an amino acid sequence or nucleotide sequence means 95% or more, preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Say.

  In the present specification, the “amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added” is 1 or more and 30 or less, preferably 1 or more and 20 or less, more preferably 1 An amino acid sequence in which 10 or less, more preferably 1 or more and 5 or less, and still more preferably 1 or more and 3 or less amino acids are deleted, inserted, substituted or added. In the present specification, the “nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added” is 1 or more and 90 or less, preferably 1 or more and 60 or less, more preferably 1 Examples include nucleotide sequences in which one or more and 30 or less, more preferably 1 or more and 15 or less, more preferably 1 or more and 10 or less nucleotides are deleted, inserted, substituted or added. In the present specification, “addition” of amino acids or nucleotides includes addition of one or more amino acids or nucleotides to one and both ends of the sequence.

  In this specification, “operable linkage” between a control region and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. Say. The procedure of “operable linkage” between a gene and a regulatory region is well known to those skilled in the art.

  In the present specification, unless otherwise specified, “upstream” and “downstream” relating to a gene refer to upstream and downstream in the transcription direction of the gene.

  In the present specification, the “corresponding position” or “corresponding region” on the amino acid sequence or nucleotide sequence represents the maximum homology between the target sequence and the reference sequence (for example, the amino acid sequence shown by SEQ ID NO: 11). It can be determined by aligning as given. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms and the procedures are known to those skilled in the art. For example, the alignment can be performed manually based on the above-described Lipman-Pearson method or the like, but the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res. 22: 4673-4680). ) With default settings. Alternatively, it is possible to use Clustal W2 or Clustal omega, which is a revised version of Clustal W. Clustal W, Clustal W2, and Clustal omega are, for example, European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) and Japanese DNA data operated by the National Institute of Genetics. It can be used on the website of the bank (DDBJ [www.ddbj.nig.ac.jp/searches-j.html]). The position or region of the target sequence aligned corresponding to an arbitrary region of the reference sequence by the above alignment is regarded as a “corresponding position” or “corresponding region” for the arbitrary region.

  In the present specification, the “adh gene” is a gene encoding alcohol dehydrogenase, and is a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1; having at least 80% identity with the nucleotide sequence represented by SEQ ID NO: 1. A polynucleotide comprising a nucleotide sequence and encoding a polypeptide having alcohol dehydrogenase activity; and a nucleotide in which one or more nucleotides have been deleted, inserted, substituted or added to the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide consisting of a sequence and having alcohol dehydrogenase activity. Examples of the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 include a gene encoding alcohol dehydrogenase 1 (ADH1) (SEQ ID NO: 2) derived from Rhizopus delmar.

  Therefore, the “adh gene” in the present specification is also a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 2 And a polynucleotide encoding a polypeptide having alcohol dehydrogenase activity; and an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted or added to the amino acid sequence shown in SEQ ID NO: 2 And a polynucleotide encoding a polypeptide having alcohol dehydrogenase activity.

  In the present specification, “reduction of alcohol dehydrogenase activity” in a mutant microorganism means that the alcohol dehydrogenase activity in the mutant microorganism is 50% or less, preferably 20% or less, more preferably 10% of the activity of the strain before the mutation (parent strain). It means that it is reduced to less than%. Microbial alcohol dehydrogenase activity is described in Applied Biochemistry and Biotechnology, 2011, Vol. 164: 1305-1322, or according to the methods described in the examples below.

  The mutant Rhizopus sp. Of the present invention is a mutant Rhizopus sp. With reduced alcohol dehydrogenase activity.

  As a parent strain of the genus Rhizopus of the present invention, filamentous fungi belonging to the genus Rhizopus, for example, Rhizopus oryzae, Rhizopus arrizus, Rhizopus chinensis, Rhizopus nigricans, Rhizopus nigricans, Rhizopus rizopus, Among these, Rhizopus oryzae and Rhizopus delmar are preferable, and Rhizopus delmar is more preferable. The mutant strain of the genus Rhizopus of the present invention can be produced by modifying the parent strain so that the alcohol dehydrogenase activity is reduced.

  Reduction of alcohol dehydrogenase activity in Rhizopus sp. Can be achieved by reducing the expression level of alcohol dehydrogenase in Rhizopus sp. Cells. For example, alcohol can be obtained by deleting or inactivating the adh gene of the genus Rhizopus, inactivating mRNA transcribed from the adh gene, or inhibiting translation of the adh gene from mRNA to protein. The activity of dehydrogenase can be reduced. Alternatively, the activity of alcohol dehydrogenase can also be reduced by reducing the activity of alcohol dehydrogenase expressed in Rhizopus sp. The above modification can be artificially performed using molecular biological or genetic engineering techniques.

  Examples of a method for inactivating mRNA of adh gene in cells include target-specific mRNA inhibition using siRNA. The basic procedure of siRNA is well known to those skilled in the art (see, for example, JP-T-2007-512808), and reagents or kits for siRNA are commercially available. A person skilled in the art can prepare a desired target-specific siRNA and inhibit the target mRNA based on known literature and manuals of commercially available products.

  Examples of the method for reducing the activity of alcohol dehydrogenase include a method using an ADH inhibitor. Examples of ADH inhibitors include 4-methylpyrazole, 1,2-diazole, 2,2,2-trifluoroethanol and the like (Alcohol, 1995, 12: 351-355, and Appl. Biochem. Biotechnol, 2011,). 164: 1305-1322 etc.).

  Methods for deleting or inactivating the adh gene in the cell include removing a part or all of the nucleotide sequence of the adh gene from the genome or replacing it with other nucleotide sequences, and others in the sequence of the adh gene. And a method of mutating the transcription or translation initiation region of the adh gene. Preferably, part or all of the nucleotide sequence of the adh gene is deleted or inactivated. More specific examples include a method for specifically deleting or inactivating the adh gene on the genome of a cell, and a random deletion or inactivating mutation in the gene in the cell, and then the adh gene And a method for selecting a cell having a desired mutation by evaluating the expression level and activity of the alcohol dehydrogenase encoded by the gene, or analyzing the gene.

As a method of randomly deleting or inactivating a gene in a cell, a DNA fragment obtained by randomly cloning the inactivated gene is introduced into the cell, and homologous recombination is performed between the gene on the genome of the cell. And a method of inducing mutations by irradiating cells with ultraviolet rays, γ rays and the like. Site-directed mutagenesis system Mutan-SuperExpress Km kit (Takara Bio), Transformer ^™ Site-Directed Mutagenesis kit (CloneOD), Site-Directed Mutagenesis System Mutan-SuperExpress Km kit Commercially available kits such as Plus-Mutageness Kit (Toyobo Co., Ltd.) can be used. By confirming the genomic sequence of a cell having a random mutation obtained by the above method, a cell in which the adh gene is deleted or inactivated can be selected. Alternatively, cells in which the adh gene is deleted or inactivated can be selected using the expression level or activity of alcohol dehydrogenase in the cell as an index.

  Examples of a method for specifically deleting or inactivating a gene in the genome include, but are not limited to, genome editing using artificial DNA nucleases or programmable nucleases. It is done.

  In a preferred embodiment, the deletion or inactivation of the Rhizopus adh gene according to the present invention is performed by genome editing of the adh locus. In the present specification, the “adh locus” refers to a region on the genome including the DNA sequence of the adh gene, its 5′-side 1000 bp region and its 3′-side 1000 bp region.

  Genome editing specifically cleaves the DNA duplex at the target locus on the genome, induces deletion, insertion, or substitution of nucleotides in the process of repairing the cleaved DNA, or inserts a foreign polynucleotide. This is a technique for site-specific modification of the genome. Such a technology is known as TALEN (transition activator-like effector nuclease), ZFN (zinc-finger nuclase), or CRISPR (Clustered Regenerative Interspaced 15). : 321-334, Nucleic Acids Research, 2011, 39: e82). Kits for genome editing based on these techniques are commercially available, and can be purchased from, for example, Life technologies, Cellectis, and Transposagen Biopharmaceuticals.

  TALEN will be described in detail for the purpose of illustration of genome editing technology only. TALEN is a protein having a transcriptional activator-like (TAL) effector derived from the DNA binding domain of the genus Xanthomonas, which is a phytopathogenic bacterium, and a DNA cleavage domain consisting of DNA nuclease Fok1. The TALEN TAL effector has a structure in which about 17 repeating sequences (repeats) consisting of about 34 amino acids are linked, and each repeat recognizes one nucleotide. The type of base that each repeat specifically recognizes depends on the type of two consecutive amino acid residues (referred to as repeat-variable residues; referred to as RVD) at a specific position in each repeat. The types of RVD amino acid residue pairs corresponding to the four bases ATGC are known. Thus, one skilled in the art can construct a TALEN that specifically recognizes the target genomic DNA sequence. In genome editing by TALEN, a set of TALENs that specifically recognize upstream and downstream sequences in the vicinity of the genomic DNA region to be cleaved are designed and expressed in cells. In general, a gene encoding a target-specific TALEN is constructed, introduced into a cell, and the TALEN is expressed in the cell. When each expressed TALEN binds to the corresponding genomic DNA, Fok1 contained therein forms a dimer on the cleavage target genomic DNA region and cleaves the DNA duplex.

  Examples of TALEN for deletion or inactivation of Rhizopus adh gene include a TALEN pair consisting of the following polypeptides (L) and (R).

  Polypeptide (L): TALEN having a TAL effector targeting a sequence represented by 5′-TGTCTGAAGAAAACTTTCT-3 ′ (SEQ ID NO: 9) in the sense strand of the adh gene, and a DNA cleavage domain comprising Fok1-like DNA nuclease Polypeptide. Preferably, the TAL effector (hereinafter also referred to as TAL effector (L)) contained in the polypeptide (L) includes a polypeptide in which 17 repeat sequences (repeats) are linked, and 5′-TGTCTGAAGAAAACTTTCT-3 ′. The sequence shown by (SEQ ID NO: 9) is recognized.

  Polypeptide (R): having a TAL effector targeting the sequence shown by 5′-TAAACTTACTCTTGGCA-3 ′ (SEQ ID NO: 10) in the antisense strand of the adh gene, and a DNA cleavage domain consisting of a Fok1-like DNA nuclease TALEN polypeptide. Preferably, the TAL effector (hereinafter also referred to as TAL effector (R)) contained in the polypeptide (R) includes a polypeptide in which 16 repetitive sequences (repeats) are linked, and 5′-TAAACTACTACTCTGGCA-3 ′. The sequence represented by (SEQ ID NO: 10) is recognized.

  Of the 17 repeats in the TAL effector (L), the 1st to 16th repeats from the upstream each consist of an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 11 Or consisting of an amino acid sequence in which 5 or less amino acid residues are deleted, inserted, substituted or added to the amino acid sequence shown in SEQ ID NO: 11. Preferably, the 1st to 16th repeats from the upstream are each deleted or inserted with 3 or less amino acid residues in the portion other than RVD described later with respect to the amino acid sequence represented by SEQ ID NO: 11 Consists of a substituted or added amino acid sequence. The 17th repeat located in the most downstream among the repeats in the TAL effector (L) consists of an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 27.

  Of the 16 repeats in the TAL effector (R), the 1st to 15th repeats from the upstream are each composed of an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 28. Or an amino acid sequence in which 5 or less amino acid residues are deleted, inserted, substituted or added to the amino acid sequence shown in SEQ ID NO: 28. Preferably, the 1st to 15th repeats from the upstream are deleted or inserted with 3 or less amino acid residues in the portion other than RVD described later with respect to the amino acid sequence represented by SEQ ID NO: 28, respectively. Consists of a substituted or added amino acid sequence. The 16th repeat located at the most downstream of the repeats in the TAL effector (R) consists of an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 43.

Preferably, the 17 repeats in the TAL effector (L) each have two amino acid residues (repeat) that are base recognition sites at positions corresponding to amino acids 12 to 13 of the amino acid sequence represented by SEQ ID NO: 11. -Variable diresidues (RVD), and the RVD of each repeat recognizes the 2nd to 18th bases of the sequence shown in SEQ ID NO: 9. Also preferably, each of the 16 repeats in the TAL effector (R) has an RVD at a position corresponding to amino acids 12 to 13 of the amino acid sequence represented by SEQ ID NO: 28, and the RVD of each repeat has the sequence Recognizes the 2nd to 17th bases of the sequence shown by No. 10. The type of amino acid residue of RVD depends on the type of target base as shown below.
Target base RVD
A NI
C HD
G / A NN
T NG

  However, as long as the TAL effector (L) can recognize the sequence represented by SEQ ID NO: 9, 2 or less, preferably 1 of the 17 repeats have RVD corresponding to SEQ ID NO: 9. Not necessary. In other words, 15 or more, preferably 16 or more of the 17 repeats may have RVD corresponding to SEQ ID NO: 9. As long as the TAL effector (R) can recognize the sequence represented by SEQ ID NO: 10, 2 or less, preferably 1 of the 16 repeats do not have an RVD corresponding to SEQ ID NO: 10. Also good. In other words, 14 or more, preferably 15 or more of the 16 repeats may have RVD corresponding to SEQ ID NO: 10.

In a further preferred embodiment, each of the 17 repeats of the TAL effector (L) is composed of the following amino acid sequences (1) to (17) in order from the upstream:
(1) the amino acid sequence represented by SEQ ID NO: 11, or an amino acid sequence having at least 95% identity with this;
(2) the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 95% identity with this;
(3) the amino acid sequence represented by SEQ ID NO: 13, or an amino acid sequence having at least 95% identity with this;
(4) the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 95% identity with this;
(5) the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 95% identity with this;
(6) the amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence having at least 95% identity with this;
(7) the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having at least 95% identity thereto;
(8) the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 95% identity with this;
(9) the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 95% identity with this;
(10) the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence having at least 95% identity thereto;
(11) the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence having at least 95% identity with this;
(12) the amino acid sequence represented by SEQ ID NO: 22, or an amino acid sequence having at least 95% identity with this;
(13) the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence having at least 95% identity thereto;
(14) the amino acid sequence represented by SEQ ID NO: 24, or an amino acid sequence having at least 95% identity thereto;
(15) the amino acid sequence represented by SEQ ID NO: 25, or an amino acid sequence having at least 95% identity thereto;
(16) the amino acid sequence represented by SEQ ID NO: 26, or an amino acid sequence having at least 95% identity thereto;
(17) The amino acid sequence represented by SEQ ID NO: 27, or an amino acid sequence having at least 95% identity thereto.

Preferably, the amino acid sequences (1) to (17) each have an RVD consisting of the following amino acid residue pairs:
(1) amino acid residue NN (guanine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 11;
(2) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 12;
(3) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 13;
(4) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 14;
(5) amino acid residue NN (guanine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 15;
(6) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 16;
(7) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 17;
(8) amino acid residue NN (guanine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 18;
(9) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 19;
(10) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 20;
(11) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 21;
(12) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 22;
(13) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 23;
(14) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 24;
(15) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 25;
(16) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 26;
(17) Amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 27.
However, as long as the TAL effector (L) can recognize the sequence represented by 5′-TGTCTGAAGAAAACTTTCT-3 ′ (SEQ ID NO: 9), 15 or more of the repeats (1) to (17) above, preferably 16 What is necessary is just to have the said RVD at least.

In another more preferred embodiment, each of the 16 repeats of the TAL effector (R) consists of the following amino acid sequences (1 ′) to (16 ′) in order from upstream:
(1 ′) the amino acid sequence represented by SEQ ID NO: 28, or an amino acid sequence having at least 95% identity thereto;
(2 ′) the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence having at least 95% identity with this;
(3 ′) the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 95% identity with this;
(4 ′) the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 95% identity with this;
(5 ′) the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence having at least 95% identity with this;
(6 ′) the amino acid sequence represented by SEQ ID NO: 33, or an amino acid sequence having at least 95% identity thereto;
(7 ′) the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence having at least 95% identity thereto;
(8 ′) the amino acid sequence shown by SEQ ID NO: 35, or an amino acid sequence having at least 95% identity with this;
(9 ′) the amino acid sequence shown by SEQ ID NO: 36, or an amino acid sequence having at least 95% identity with this;
(10 ′) the amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 95% identity thereto;
(11 ′) the amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 95% identity thereto;
(12 ′) the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 95% identity with this;
(13 ′) the amino acid sequence shown by SEQ ID NO: 40, or an amino acid sequence having at least 95% identity with this;
(14 ′) the amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 95% identity thereto;
(15 ′) the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 95% identity with this;
(16 ′) An amino acid sequence represented by SEQ ID NO: 43 or an amino acid sequence having at least 95% identity thereto.

Preferably, the amino acid sequences (1 ′) to (16 ′) each have an RVD consisting of the following amino acid residue pairs:
(1 ′) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 28;
(2 ′) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 29;
(3 ′) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 30;
(4 ′) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 31;
(5 ′) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 32;
(6 ′) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 33;
(7 ′) amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 34;
(8 ′) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 35;
(9 ′) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 36;
(10 ′) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 37;
(11 ′) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 38;
(12 ′) amino acid residue NG (thymine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 39;
(13 ′) amino acid residue NN (guanine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 40;
(14 ′) amino acid residue NN (guanine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 41;
(15 ′) amino acid residue HD (cytosine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 42;
(16 ′) An amino acid residue NI (adenine recognition site) at a position corresponding to amino acids 12 to 13 of SEQ ID NO: 43.
However, as long as the TAL effector (R) can recognize the sequence represented by 5′-TAAACTTACTCTTGGCA-3 ′ (SEQ ID NO: 10), preferably 14 or more of the repeats (1 ′) to (16 ′) above, It is sufficient that 15 or more have the RVD.

  The Fok1-like DNA nuclease contained in the polypeptides (L) and (R) refers to a nuclease that dimerizes and functions as a DNA endonuclease. Preferably, the Fok1-like DNA nuclease is encoded by a polynucleotide consisting of a sequence represented by nucleotides 2416 to 3006 of SEQ ID NO: 3, or a sequence consisting of at least 95% of the same and dimerized to form a DNA endonuclease. It is a nuclease that functions as a nuclease.

Preferred examples of the polypeptides (L) and (R) include the following:
Polypeptide (L):
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4; or the TAL comprising the amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 4 and targeting the sequence represented by SEQ ID NO: 9 A polypeptide having an effector (L) and a DNA cleavage domain comprising the Fok1-like DNA nuclease,
Polypeptide (R):
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6, or the TAL comprising the amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 6 and targeting the sequence represented by SEQ ID NO: 10 A polypeptide having an effector (R) and a DNA cleavage domain comprising the Fok1-like DNA nuclease.

Other preferred examples of the polypeptides (L) and (R) include polypeptides encoded by the following polynucleotides (l) and (r), respectively:
Polynucleotide (l):
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3; or the TAL comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 3 and targeting the sequence represented by SEQ ID NO: 9 A polynucleotide encoding a polypeptide having an effector (L) and a DNA cleavage domain comprising the Fok1-like DNA nuclease,
Polynucleotide (r):
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5; or the TAL comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 5 and targeting the sequence represented by SEQ ID NO: 10 A polynucleotide encoding a polypeptide having an effector (R) and a DNA cleavage domain comprising the Fok1-like DNA nuclease.

  In a preferred embodiment, the polypeptide (L) is a polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 4 or a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 3. In a preferred embodiment, the polypeptide (R) is a polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 6 or a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 5.

  TALEN comprising the polypeptides (L) and (R) specifically binds to the sense strand and the antisense strand of DNA (SEQ ID NO: 68) containing the adh locus on the Rhizopus genome, The DNA at the locus is cleaved, and the adh gene is deleted or inactivated.

  Therefore, in a preferred embodiment, the above-mentioned polypeptides (L) and (R) or a polynucleotide encoding them is introduced into the parent strain of Rhizopus, so that the adh gene of Rhizopus is deleted or not present. Activated to produce the mutant Rhizopus spp. Of the present invention. Preferable examples of the polynucleotide encoding the polypeptides (L) and (R) include the above-described polynucleotides (l) and (r), respectively.

  Expression of a foreign exonuclease together with TALEN in the cell promotes destruction of TALEN target DNA (Scientific Reports, 2013, 3: 1253, DOI: 10.1038 / srep01253, Nat Methods, 2012, 9: 973 -975). Therefore, in a preferred embodiment of the present invention, in addition to the TALEN peptide or a polynucleotide encoding the same, an exonuclease or a polynucleotide encoding the same is further introduced into the Rhizopus sp. Parent strain. The exonuclease is not particularly limited as long as it is an exonuclease derived from Rhizopus, but preferably includes those belonging to exonuclease 1 or exonuclease 2, more preferably derived from a genus Rhizopus, such as Rhizopus oryzae or Rhizopus. Examples include delmar-derived exonuclease.

  More preferable examples of the exonuclease include Rhizopus oryzae-derived exonuclease (SEQ ID NO: 8) encoded by a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 7 or 69. Therefore, as a more preferred example of the exonuclease used in the present invention, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8; an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 8 A polypeptide encoded by a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 7; a nucleotide sequence having at least 95% identity with the nucleotide sequence set forth in SEQ ID NO: 7 A polypeptide having an exonuclease activity encoded by the polynucleotide; a polypeptide encoded by the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 69; and at least the nucleotide sequence represented by SEQ ID NO: 69 Polypeptide comprising the polynucleotide-encoded exonuclease activity consisting of the nucleotide sequence having 95% identity, and the like.

  For expression of TALEN or exonuclease in the cell, a TALEN or exonuclease peptide or a polynucleotide encoding the peptide may be introduced into the cell. Preferably, an expression vector containing a polynucleotide encoding each TALEN or exonuclease is introduced into the cell, and the TALEN or exonuclease is expressed in the cell. Each TALEN and exonuclease polynucleotide may be contained in the same vector, but may be contained in separate vectors. The expression vector is not particularly limited as long as it is stably retained in the cell into which it is introduced and can be propagated. Examples of expression vectors suitable for Rhizopus sp. Include pUC18 / 19, pUC118 / 119, pBR322, pMW218 / 219, pPTR1 / 2 (TaKaRa), pRI909 / 910 (TaKaRa), and pDJB2 (DJ Ballance et al. , Gene, 36, 321-331, 1985), pAB4-1 (van Hartingsveld W et al., Mol Gen Genet, 206, 71-75, 1987), pLeu4 (MIG Roncero et al., Gene). 84, 335-343, 1989), pPyr225 (CD Skory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et a. ., Curr Genet, 18,447-451,1990), and the like.

  For efficient expression of TALEN in cells, the polynucleotide encoding TALEN or exonuclease is preferably operably linked to a promoter working in Rhizopus. Examples of promoters working in Rhizopus sp. Include an ldhA promoter (US Pat. No. 6,268,189), a pgk1 promoter (International Publication No. 2001/73083), a pgk2 promoter (International Publication No. 2001/72967), a pdcA promoter and an amyA promoter. (Archives of Microbiology, 2006, 186: 41-50), tef and 18S rRNA promoters (US Patent Application Publication No. 2010/112651), and adh1 promoter (Japanese Patent Application No. 2015-155759), but are not limited thereto.

  The polynucleotide encoding TALEN or exonuclease or an expression vector containing the same can be obtained by a general transformation method, for example, electroporation method, transformation method, transfection method, conjugation method, protoplast method, particle gun method, agro It can be introduced into cells using a bacterial method or the like.

  Cells in which the target gene has been deleted or inactivated by the introduction of TALEN can be selected based on confirmation of the genomic sequence of the cells, or the expression level or activity of alcohol dehydrogenase, as in the case of random mutation.

  By the above procedure, the mutant Rhizopus sp. Of the present invention with reduced alcohol dehydrogenase activity can be produced. The mutant Rhizopus spp. Of the present invention has improved organic acid productivity as compared to the strain (parent strain) before the mutation. Therefore, when the mutant Rhizopus bacterium of the present invention is cultured, an organic acid can be produced efficiently. Therefore, the present invention provides a method for producing an organic acid, comprising culturing the mutant Rhizopus spp. Of the present invention. Preferred examples of the organic acid include lactic acid, fumaric acid, succinic acid, malic acid and α-ketoglutaric acid.

  Production of the organic acid using the mutant genus Rhizopus of the present invention is carried out by culturing the mutant genus Rhizopus of the present invention in accordance with a general method for culturing Rhizopus, and then recovering the organic acid from the culture be able to. For example, an organic acid can be produced by culturing the mutant Rhizopus spp. Of the present invention for several days in an appropriate medium at 10 ° C. to 50 ° C., preferably 25 ° C. to 35 ° C. The culture period may be a period in which the target organic acid is sufficiently produced. For example, if the target organic acid is lactic acid, lactic acid is produced by culturing at the above-mentioned temperature for 1 to 168 hours, preferably 2 to 72 hours, more preferably 4 to 24 hours. Alternatively, if the target organic acid is fumaric acid, fumaric acid is produced by culturing at the above-mentioned temperature for 1 to 168 hours, preferably 2 to 72 hours, more preferably 4 to 36 hours. The same applies to succinic acid, malic acid, and α-ketoglutaric acid.

  The medium for culturing the mutant Rhizopus spp. Of the present invention is not particularly limited as long as Rhizopus spp. Can grow healthy and produce the desired organic acid. Examples of the medium include monosaccharides such as glucose and xylose; oligosaccharides such as sucrose, lactose and maltose; solid medium or liquid medium using a polysaccharide such as starch as a substrate, or commercially available PDB medium (potato dextrose medium; Becton Dickinson and Company, etc.), PDA medium (Becton Dickinson and Company, etc.), LB medium (Luria-Bertani medium; trade name “Daigo”, etc.), NB medium (Nutient Broth; Becton, etc.) -Dickinson & Company, etc.), SB medium (Sabouraud medium; manufactured by OXOID, etc.) can be used. Furthermore, the medium contains biological components such as glycerin and citric acid; nitrogen sources such as ammonium salts, nitrates, nitrites, urea, amino acids, natural nitrogen sources such as soybean peptides; sodium, potassium, magnesium, zinc, iron, Various salts such as phosphoric acid can be added as appropriate.

  After the culture, an organic acid can be obtained by collecting the culture supernatant from the medium. If necessary, the organic acid in the culture solution can be converted into an organic acid salt by a gradient method, membrane separation, centrifugation, electrodialysis method, use of ion exchange resin, distillation, salting-out, or a combination thereof. After the recovery, the organic acid may be isolated or purified from the recovered organic acid salt.

  In one embodiment, in order to produce an organic acid more efficiently, production may be performed by the following steps. That is, a spore suspension of the mutated Rhizopus spp. Of the present invention is prepared (step A), which is cultured in a culture solution to germinate spores to prepare a mycelium (step B1), and preferably further the mycelium. An organic acid can be efficiently produced by growing the body (step B2) and then culturing the prepared mycelium to produce an organic acid (step C).

<Step A: Preparation of spore suspension>
The spores of the genus Rhizopus of the present invention can be obtained, for example, by using an inorganic agar medium (composition example: 2% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate / 7 hydrate). , 0.009% zinc sulfate heptahydrate, 1.5% agar, each concentration is% (w / v)), PDA medium, etc., and 10 to 40 ° C., preferably 27 A spore suspension can be prepared by forming a spore by performing stationary culture at -30 ° C for 7 to 10 days and suspending in a physiological saline or the like. The mycelium may or may not be contained in the spore suspension.

<Step B1: Preparation of mycelium>
The spore suspension obtained in step A is inoculated into a culture solution and cultured, and the spores are germinated to obtain mycelium. The number of filamentous fungi inoculated into the culture solution is 1 × 10 ² to 1 × 10 ⁸ spores / mL-culture solution, preferably 1 × 10 ^{2 to} 5 × 10 ⁴ spores / mL-culture solution, More preferably, it is 5 * 10 < ² > -1 * 10 < ⁴ > spores / mL-culture liquid, More preferably, it is 1 * 10 < ³ > -1 * 10 < ⁴ > spores / mL-culture liquid.

  A commercially available medium such as a PDB medium, LB medium, NB medium, or SB medium can be used as the culture solution for spore germination used in this step. In addition, from the viewpoints of germination rate and cell growth, the culture solution may include monosaccharides such as glucose and xylose, oligosaccharides such as sucrose, lactose, and maltose, or polysaccharides such as starch as a carbon source; glycerin, citric acid Biological components such as ammonium sources, ammonium salts, nitrates, nitrites, urea, etc., amino acids, natural nitrogen sources such as soybean peptides, etc .; other inorganic substances such as sodium, potassium, magnesium, zinc, iron, phosphoric acid, etc. Can be added as appropriate. The preferred concentration of monosaccharide, oligosaccharide, polysaccharide and glycerin is 0.1-30% (w / v), the preferred concentration of citric acid is 0.01-10% (w / v), and the preferred concentration of nitrogen source is 0 0.01 to 1% (w / v), and the preferred concentration of the inorganic substance is 0.0001 to 0.5% (w / v).

  In step B1, the mutant Rhizopus sp. Of the present invention is cultured using the culture solution. The culture may be performed according to a normal procedure. For example, a fungal spore is inoculated into a culture vessel containing a culture solution, and preferably stirred at a temperature of 25 to 42.5 ° C. with stirring at 80 to 250 rpm, more preferably 100 to 170 rpm, preferably 24. Incubate for ~ 120 hours, more preferably 48-72 hours. The amount of the culture solution used for the culture may be appropriately adjusted according to the culture container. For example, in the case of a flask with a 200 mL baffle, it may be about 100 to 300 mL in the case of a flask with a 500 mL baffle. That's fine. By this culture, filamentous fungal spores germinate and grow into mycelium.

<Step B2: Growth of mycelium>
From the viewpoint of improving organic acid production ability, it is preferable to perform a step (step B2) of further culturing and growing the mycelium obtained in step B1. The growth culture medium used in step B2 is not particularly limited, and may be any inorganic culture liquid containing glucose that is normally used. For example, 7.5-30% glucose, 0.05-2% ammonium sulfate, 0 0.03 to 0.6% potassium dihydrogen phosphate, 0.01 to 0.1% magnesium sulfate heptahydrate, 0.005 to 0.1% zinc sulfate heptahydrate, and 3.75 to Examples thereof include a culture solution containing 20% calcium carbonate (both concentrations are% (w / v)), preferably 10% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0 A culture solution containing 0.025% magnesium sulfate.7 hydrate, 0.009% zinc sulfate.7 hydrate, and 5.0% calcium carbonate (the concentration is% (w / v)). . The amount of the culture solution may be appropriately adjusted according to the culture vessel. For example, in the case of a 500 mL Erlenmeyer flask, it may be 50 to 300 mL, preferably 100 to 200 mL. The bacterial cells cultured in step B1 are inoculated into this culture solution in a wet weight of 1 to 20 g-bacteria / 100 mL-medium, preferably 3 to 10 g-bacteria / 100 mL-medium, 100 to 300 rpm, The culture is performed for 12 to 120 hours, preferably for 16 to 72 hours under a culture temperature control of 25 to 42.5 ° C. while preferably stirring at 170 to 230 rpm.

<Process C: Organic acid production>
The mycelium of the filamentous fungus obtained in the above procedure (B1 or B2) can be cultured to cause the fungus to produce an organic acid, and then the produced organic acid can be recovered.
The culture solution for organic acid production used in the step (C) includes a carbon source such as glucose, a nitrogen source such as ammonium salt, various metal salts, and the like, and may be any culture solution that can produce an organic acid. Examples of the culture solution used in the step (C) include 7.5-30% glucose, 0.05-2% ammonium sulfate, 0.03-0.6% potassium dihydrogen phosphate, 0.01- 0.1% magnesium sulfate 7-hydrate, 0.005-0.1% zinc sulfate 7-hydrate, and 3.75-20% calcium carbonate (all concentrations are% (w / v)) Examples of the culture medium include 10 to 12.5% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, and the like. Examples include a culture solution containing 009% zinc sulfate heptahydrate and 5.0% calcium carbonate (both concentrations are% (w / v)).

  The amount of the culture solution used in the step (C) may be appropriately adjusted according to the culture vessel. For example, in the case of a 200 mL Erlenmeyer flask, about 20 to 80 mL, and in the case of a 500 mL Erlenmeyer flask, about 50 to 200 mL. If it is. In this culture solution, the cells obtained in Step B1 or B2 are in a wet weight of 5 g to 90 g-bacteria / 100 mL-culture solution, preferably 5 g to 50 g-bacteria / 100 mL-culture solution. And incubating at 100 to 300 rpm, preferably 170 to 230 rpm, under a culture temperature control of 25 to 45 ° C. for 2 hours to 72 hours, preferably 4 hours to 36 hours.

  As exemplary embodiments of the present invention, the following materials, manufacturing methods, uses, or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[1] A mutant Rhizopus genus having reduced activity of alcohol dehydrogenase.

[2] The mutant Rhizopus spp. Described in [1], wherein the adh gene is preferably deleted or inactivated.

[3] Preferably, the adh gene is:
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
A polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity; and
A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity;
The mutant Rhizopus genus according to [2], which is at least one selected from the group consisting of:

[4] Preferably, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence having at least 95% identity thereto, and the nucleotide sequence represented by SEQ ID NO: 5, or at least 95% thereof The mutant Rhizopus genus according to [2] or [3], wherein a polynucleotide comprising a nucleotide sequence having identity is introduced.

[5] Preferably, the mutated Rhizopus spp. According to [4], into which an exonuclease or a polynucleotide encoding the same is further introduced.

[6] The exonuclease is
Preferably, it is an exonuclease derived from the genus Rhizopus,
More preferably, it is an exonuclease derived from Rhizopus oryzae or Rhizopus delmar.
[5] The mutant Rhizopus sp.

[7] Preferably, the polynucleotide encoding the exonuclease is
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 7 and encoding a polypeptide having exonuclease activity;
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 69;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 69 and encoding a polypeptide having exonuclease activity;
A polynucleotide encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8; or a polypeptide comprising an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 8 and having exonuclease activity The encoding polynucleotide,
The mutated Rhizopus spp. According to [5].

[8] The activity of alcohol dehydrogenase is preferably reduced to 50% or less, more preferably 20% or less, and even more preferably 10% or less compared to that before mutation, any one of [1] to [7] 2. The mutant Rhizopus sp.

[9] The Rhizopus sp.
Rhizopus oryzae or Rhizopus delmar is preferred,
More preferably Rhizopus delmar,
The mutant Rhizopus genus according to any one of [1] to [8].

[10] The mutant Rhizopus bacterium according to any one of [1] to [9], wherein organic acid productivity is improved.

[11] A method for producing a mutant Rhizopus, comprising reducing the activity of alcohol dehydrogenase in Rhizopus.

[12] A method for improving organic acid productivity of Rhizopus sp., Comprising reducing the activity of alcohol dehydrogenase in Rhizopus sp.

[13] The method according to [11] or [12], preferably comprising deleting or inactivating the adh gene in Rhizopus sp.

[14] Preferably, the adh gene is the following:
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
A polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity; and
A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity;
The method according to [13], which is at least one selected from the group consisting of:

[15] The method according to [13] or [14], wherein the deletion or inactivation of the adh gene is preferably performed by genome editing of the adh gene locus using an artificial DNA cleaving enzyme.

[16] The method according to [15], wherein the genome editing is preferably performed by TALEN, Crispr-cas9 system, or ZFN.

[17] The genome editing
Preferably, a TALEN peptide or a polynucleotide encoding the same is introduced into the Rhizopus sp.
More preferably, a polynucleotide encoding TALEN is introduced into the Rhizopus sp.
[16] The method described in the above.

[18] Preferably, the TALEN comprises the following polypeptides (L) and (R):
Polypeptide (L):
Has a TAL effector with 17 repeats linked;
Among the 17 repeats, the 1st to 16th repeats from the upstream are each an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 11, or the amino acid sequence represented by SEQ ID NO: 11 Consisting of an amino acid sequence in which no more than 5 amino acid residues are deleted, inserted, substituted or added to
The 17th repeat from the upstream of the 17 repeats consists of an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 27;
The TAL effector recognizes the sequence represented by SEQ ID NO: 9.
Polypeptide (R):
Has a TAL effector with 16 repeats linked;
Among the 16 repeats, the 1st to 15th repeats from the upstream are each an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 28, or the amino acid sequence represented by SEQ ID NO: 28 Consisting of an amino acid sequence in which no more than 5 amino acid residues are deleted, inserted, substituted or added to
The 16th repeat from the upstream of the 16 repeats consists of an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 43;
The TAL effector recognizes the sequence represented by SEQ ID NO: 10.

[19] Preferably, the 17 repeats in the TAL effector of the polypeptide (L) are each repeat-variable residues (RVD) at positions corresponding to amino acids 12 to 13 of the amino acid sequence represented by SEQ ID NO: 11. The RVD of each repeat recognizes the 2nd to 18th bases of the sequence represented by SEQ ID NO: 9, and the method according to [18].

[20] Preferably, the 16 repeats in the TAL effector of the polypeptide (R) are each represented by repeat-variable residues (RVD) at positions corresponding to amino acids 12 to 13 of the amino acid sequence shown by SEQ ID NO: 28. And the RVD of each repeat recognizes the 2nd to 17th bases of the sequence represented by SEQ ID NO: 10.

[21] The method according to [18], wherein the 17 repeats in the TAL effector of the polypeptide (L) are composed of the following amino acid sequences (1) to (17) in order from the upstream.
(1) the amino acid sequence represented by SEQ ID NO: 11, or the amino acid sequence having at least 95% identity thereto (2) amino acid sequence represented by SEQ ID NO: 12, or the amino acid sequence having at least 95% identity thereto (3) The amino acid sequence shown by SEQ ID NO: 13, or the amino acid sequence having at least 95% identity with this (4) The amino acid sequence shown by SEQ ID NO: 14, or the amino acid sequence having at least 95% identity with this (5) The amino acid sequence shown by SEQ ID NO: 15, or the amino acid sequence having at least 95% identity with this (6) The amino acid sequence shown by SEQ ID NO: 16, or the amino acid sequence having at least 95% identity with this (7) the amino acid sequence shown in SEQ ID NO: 17, or an amino acid having at least 95% identity thereto Sequence (8) Amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having at least 95% identity thereto (9) Amino acid sequence represented by SEQ ID NO: 19, or an amino acid having at least 95% identity thereto Sequence (10) Amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence having at least 95% identity thereto (11) Amino acid sequence represented by SEQ ID NO: 21, or an amino acid having at least 95% identity thereto Sequence (12) Amino acid sequence represented by SEQ ID NO: 22, or an amino acid sequence having at least 95% identity thereto (13) Amino acid sequence represented by SEQ ID NO: 23, or amino acids having at least 95% identity thereto Sequence (14) amino acid sequence set forth in SEQ ID NO: 24, or at least 95% identical thereto (15) the amino acid sequence shown in SEQ ID NO: 25, or the amino acid sequence shown in SEQ ID NO: 26 having at least 95% identity thereto, or the amino acid sequence shown in SEQ ID NO: 26, or at least 95% identity therewith (17) the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 95% identity thereto

[22] The method according to [21], wherein preferably 15 or more, more preferably 16 or more of the sequences (1) to (17) have the following amino acid residues.
Regarding (1): amino acid residue NN at a position corresponding to amino acids 12-13 of SEQ ID NO: 11
Regarding (2): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 12
Regarding (3): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 13
Regarding (4): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 14
Regarding (5): amino acid residue NN at the position corresponding to amino acids 12-13 of SEQ ID NO: 15
Regarding (6): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 16
Regarding (7): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 17
Regarding (8): amino acid residue NN at the position corresponding to amino acids 12-13 of SEQ ID NO: 18
Regarding (9): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 19
Regarding (10): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 20
Regarding (11): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 21
Regarding (12): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 22
Regarding (13): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 23
Regarding (14): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 24
Regarding (15): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 25
Regarding (16): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 26
Regarding (17): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 27

[23] The method according to [18], wherein the 16 repeats in the TAL effector of the polypeptide (R) are preferably composed of the following amino acid sequences (1 ′) to (16 ′) in order from upstream: .
(1 ′) the amino acid sequence shown by SEQ ID NO: 28, or the amino acid sequence having at least 95% identity with this (2 ′) amino acid sequence shown by SEQ ID NO: 29, or at least 95% identity with this Amino acid sequence (3 ′) amino acid sequence represented by SEQ ID NO: 30, or amino acid sequence having at least 95% identity with this (4 ′) amino acid sequence represented by SEQ ID NO: 31, or at least 95% identity with this An amino acid sequence (5 ′) having the amino acid sequence represented by SEQ ID NO: 32, or an amino acid sequence having at least 95% identity thereto (6 ′) an amino acid sequence represented by SEQ ID NO: 33, or at least 95% Amino acid sequence having identity (7 ′) The amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 95% identity thereto Amino acid sequence (8 ′) amino acid sequence represented by SEQ ID NO: 35, or an amino acid sequence having at least 95% identity with this (9 ′) amino acid sequence represented by SEQ ID NO: 36, or at least 95% identical thereto Amino acid sequence (10 ′) having amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 95% identity thereto (11 ′) amino acid sequence represented by SEQ ID NO: 38, or at least 95% thereof The amino acid sequence shown in SEQ ID NO: 39, or the amino acid sequence shown in SEQ ID NO: 40 having at least 95% identity (13 ′), the amino acid sequence shown in SEQ ID NO: 40, or at least Amino acid sequence having 95% identity (14 ′) amino acid sequence set forth in SEQ ID NO: 41, or at least 95 Amino acid sequence having 15% identity (15 ′) amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 95% identity (16 ′) amino acid sequence represented by SEQ ID NO: 43, or Amino acid sequence having at least 95% identity

[24] The method according to [23], wherein preferably 14 or more, more preferably 15 or more of the sequences (1 ′) to (16 ′) have the following amino acid residues.
Regarding (1 ′): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 28
Regarding (2 ′): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 29
Regarding (3 ′): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 30
Regarding (4 ′): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 31
Regarding (5 ′): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 32
Regarding (6 ′): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 33
Regarding (7 ′): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 34
Regarding (8 ′): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 35
Regarding (9 ′): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 36
Regarding (10 ′): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 37
Regarding (11 ′): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 38
Regarding (12 ′): amino acid residue NG at a position corresponding to amino acids 12-13 of SEQ ID NO: 39
Regarding (13 ′): amino acid residue NN at a position corresponding to amino acids 12-13 of SEQ ID NO: 40
Regarding (14 ′): amino acid residue NN at the position corresponding to amino acids 12-13 of SEQ ID NO: 41
Regarding (15 ′): amino acid residue HD at a position corresponding to amino acids 12-13 of SEQ ID NO: 42
Regarding (16 ′): amino acid residue NI at a position corresponding to amino acids 12-13 of SEQ ID NO: 43

[25] The method according to any one of [18] to [24], wherein the polypeptide (L) is preferably the following polypeptide:
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
It has an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 4 and has a TAL effector targeting the sequence shown in SEQ ID NO: 9 and a DNA cleavage domain consisting of a Fok1-like DNA nuclease A polypeptide;
A polypeptide encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3; or a polynucleotide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 3, and SEQ ID NO: A polypeptide having a TAL effector targeting the sequence represented by 9 and a DNA cleavage domain comprising a Fok1-like DNA nuclease.

[26] The method according to any one of [18] to [25], wherein the polypeptide (R) is preferably the following polypeptide:
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6;
It has an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 6 and has a TAL effector targeting the sequence shown in SEQ ID NO: 10 and a DNA cleavage domain consisting of a Fok1-like DNA nuclease A polypeptide;
A polypeptide encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5; or a polynucleotide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 5, and SEQ ID NO: A polypeptide having a TAL effector targeting the sequence represented by 10 and a DNA cleavage domain comprising a Fok1-like DNA nuclease.

[27] The method according to [17], wherein the polynucleotide encoding TALEN is preferably the following i) and ii):
i) A nucleotide sequence represented by SEQ ID NO: 3, or a polynucleotide comprising a nucleotide sequence having at least 95% identity thereto, or an amino acid sequence represented by SEQ ID NO: 4, or at least 95% identity thereto A polynucleotide encoding a polypeptide comprising an amino acid sequence, and
ii) a nucleotide sequence represented by SEQ ID NO: 5 or a polynucleotide comprising a nucleotide sequence having at least 95% identity thereto, or an amino acid sequence represented by SEQ ID NO: 6, or at least 95% identity thereto Polynucleotide encoding polypeptide comprising amino acid sequence

[28] Preferably, the method according to any one of [17] to [27], further comprising introducing exonuclease or a polynucleotide encoding the same into the genus Rhizopus.

[29] The exonuclease is
Preferably, it is an exonuclease derived from the genus Rhizopus,
More preferably, it is an exonuclease derived from Rhizopus oryzae or Rhizopus delmar.
[28] The method described.

[30] Preferably, the polynucleotide encoding the exonuclease is
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 7 and encoding a polypeptide having exonuclease activity;
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 69;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 69 and encoding a polypeptide having exonuclease activity;
A polynucleotide encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8; or a polypeptide comprising an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 8 and having exonuclease activity The encoding polynucleotide,
The method according to [28].

[31] The Rhizopus sp.
Rhizopus oryzae or Rhizopus delmar is preferred,
More preferably Rhizopus delmar,
[11] The method according to any one of [31] to [31].

[32] Any of [11] to [31], wherein the activity of alcohol dehydrogenase in the genus Rhizopus is reduced to preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less. The method according to claim 1.

[33] A method for producing an organic acid, comprising culturing the mutant Rhizopus bacterium according to any one of [1] to [10].

[34] The method for producing an organic acid according to [33], preferably further comprising recovering the organic acid from the culture after culturing.

[35] The method for producing an organic acid according to [33] or [34], wherein the organic acid is preferably fumaric acid, lactic acid, succinic acid, malic acid or α-ketoglutaric acid.

[36] Use of the mutant Rhizopus genus according to any one of [1] to [10] for the production of an organic acid.

[37] The use according to [36], wherein the organic acid is preferably fumaric acid, lactic acid, succinic acid, malic acid or α-ketoglutaric acid.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Example 1 Production of mutant Rhizopus (1) Production of tryptophan auxotrophic strain A tryptophan auxotrophic strain derived from Rhizopus delmar JCM (Japan Collection of Microorganisms / RIKEN) 5557 strain (hereinafter referred to as 5557 strain) Used as a parent strain for gene deletion. The tryptophan auxotrophic strain was selected and obtained from among the mutant-introduced strains obtained by ion beam irradiation of 5557 strain. Ion beam irradiation was performed at the ion irradiation facility (TIARA) of the Japan Atomic Energy Agency / Takasaki Quantum Application Laboratory. Irradiation was accelerated at ¹² C ⁵ + using an AVF cyclotron, and irradiated at 100 to 1250 Gray at an energy of 220 MeV. Spores were collected from the irradiated cells, and Rhizopus delmar 02T6 strain having a 1 base deletion mutation in the trpC gene region and showing tryptophan auxotrophy was obtained. This strain was used as the parent strain of the mutant Rhizopus in the following examples.

(2) Preparation of plasmid vector A DNA fragment of the trpC gene was synthesized by PCR using primers 5J5162 (SEQ ID NO: 44) and oJK163 (SEQ ID NO: 45) using 5557 strain genomic DNA as a template. Next, the DNA fragment was amplified by PCR using the plasmid pUC18 as a template and primers oJK164 (SEQ ID NO: 46) and oJK165 (SEQ ID NO: 47). The above two fragments were ligated using In-Fusion HD Cloning Kit (Clontech) to prepare plasmid pUC18-trpC.

  Next, using the genomic DNA of 5557 strain as a template, the promoter fragment and terminator fragment of adh1 were used as primers oJK202 (SEQ ID NO: 48) and oJK204 (SEQ ID NO: 49), and oJK205 (SEQ ID NO: 50) and oJK216 (SEQ ID NO: 51), respectively. Amplified by PCR using Next, using the plasmid pUC18-trpC constructed as described above as a template, the DNA fragment was amplified by PCR using primers oJK210 (SEQ ID NO: 52) and oJK211 (SEQ ID NO: 53). The above three fragments were ligated using In-Fusion HD Cloning Kit (Clontech) to prepare plasmid pUC18-trpC-Padh-Tadh. In the obtained plasmid, an adh1 promoter and a terminator are sequentially arranged downstream of the trpC gene region.

(3) Production of TALEN for adh1 gene disruption We asked Transposagen Biopharmaceuticals to produce Custom XTN TALEN (trade name of TALEN provided by Transposagen Biopharmaceuticals). This is a kit for TALEN that targets a gene encoding alcohol dehydrogenase 1 (ADH1) (adh1 gene; SEQ ID NO: 1), and includes two polynucleotides LeftTALEN-adh (SEQ ID NO: 3) and RightTALEN-adh ( It contains SEQ ID NO: 5) and binds to a region containing the adh1 gene (SEQ ID NO: 68). LeftTALEN-adh encodes TALEN that targets the sequence of 5′-TGTCTGGAAGAAACTTTCT-3 ′ (SEQ ID NO: 9) in the sense strand of the adh1 gene, and RightTALEN-adh is the 5′-TAACTACTACTCTTGGCA- in the antisense strand. It encodes a TALEN that targets the 3 '(SEQ ID NO: 10) sequence.

  Each of the polynucleotides encoding the above TALEN was prepared by the method described in (2) above. The vector was inserted into the expression vector for delmar pUC18-trpC-Pad-Tadh to express TALEN under the control of the adh1 promoter and adh1 terminator. Specifically, the vector fragment was amplified by PCR using pUC18-trpC-Padh-Tadh as a template and primers adhpro-R (SEQ ID NO: 54) and adhter-F (SEQ ID NO: 55). Subsequently, the LeftTALEN-adh fragment was amplified by PCR using the primers TALPRON-adh as a template and primers adhpro-TALEN-F (SEQ ID NO: 56) and TALEN-adhter-R (SEQ ID NO: 57). In the two fragments, there is an overlapping region of 15 bases. These two fragments were ligated with In-Fusion HD cloning kit (Clontech) to obtain a plasmid padh-LeftTALEN-adh containing LeftTALEN-adh. Similarly, the RightTALEN-adh fragment was amplified by PCR using RightTALEN-adh as a template and primers adhpro-TALEN-F (SEQ ID NO: 56) and TALEN-adhter-R (SEQ ID NO: 57), and ligated with the vector fragment. As a result, plasmid padh-RightTALEN-adh containing RightTALEN-adh was obtained.

(4) Preparation of exonuclease expression vector pUC18 vector fragment by PCR using plasmid pUC18 (Takara Bio) as a template and primers pPTR1-sal1-F (SEQ ID NO: 58) and pPTR1-sal1-R (SEQ ID NO: 59) Was amplified. In addition, using a purified genomic solution of Rhizopus oryzae NRBC5384 strain (hereinafter referred to as 5384 strain) as a template, using primers sal1-ldhpro-F3 (SEQ ID NO: 60) and ldhpro-R (SEQ ID NO: 61), Using the primers ldhpro-exo1-F2 (SEQ ID NO: 62) and exo1-pdcter-R2 (SEQ ID NO: 63), the exonuclease gene fragment (SEQ ID NO: 69) and the primers pdcTer-F (SEQ ID NO: 64) and pdcTer-sall Each pdc terminator fragment was amplified using -R (SEQ ID NO: 65). The 4 fragments thus amplified were ligated with an In-Fusion HD cloning kit (Clontech) to prepare a plasmid pldh-exo1.

(5) Gene Introduction by Particle Gunn The plasmid padh-LeftTALEN-adh and padh-RightTALEN-adh prepared in (3) above are the restriction enzyme ScaI and the plasmid pldh-exo1 prepared in (4) above is the restriction enzyme Pst1. And a DNA solution (1 μg / μL) was prepared by mixing at a concentration of 1: 1: 1. 10 μL of the DNA solution was added to 100 μL of a gold particle solution (60 mg / mL, INBIO GOLD, particle diameter 1 μm) and mixed. Furthermore, 40 μL of 0.1 M spermidine was added and stirred well by vortexing. 100 μL of 2.5 M CaCl ₂ was added and stirred for 1 minute by vortexing, then centrifuged at 6000 rpm for 30 seconds, and the supernatant was removed. After adding 200 μL of 70% EtOH to the resulting precipitate and stirring by vortex for 30 seconds, the mixture was centrifuged at 6000 rpm for 30 seconds to remove the supernatant. The resulting precipitate was resuspended with 100 μL of 100% EtOH.

  The spore of 02T6 strain obtained in (1) above was transfected with GDS-80 (Neppagene) using the above DNA-gold particle solution. The spore after gene transfer is an inorganic agar medium (20 g / L glucose, 1 g / L ammonium sulfate, 0.6 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.09 g / L (Zinc sulfate heptahydrate, 15 g / L agar), and statically cultured at 30 ° C. for about one week.

(6) Selection of adh gene-deficient strain Spores were collected from the cells cultured in (5) above, and a tryptophan-added inorganic agar medium (20 g / L glucose, 1 g / L ammonium sulfate, 0.6 g / L phosphorus) adjusted to pH 3 was used. Potassium dihydrogen acid, 0.25 g / L magnesium sulfate.7 hydrate, 0.09 g / L zinc sulfate.7 hydrate, 0.002% tryptophan, 15 g / L agar). Part of the mycelium of the grown strain was scraped with a toothpick, suspended in 10 mM Tris-HCl (pH 8.5), and incubated at 95 ° C. for 10 minutes. Thereafter, the solution was appropriately diluted with 10 mM Tris-HCl (pH 8.5) to obtain a genome template solution for colony PCR. Colony PCR was performed using the genomic template solution, primers adh-up (SEQ ID NO: 66) and adh-down (SEQ ID NO: 67), and KOD FX Neo (TOYOBO). A strain in which the amplification band length was shifted compared to the 02T6 strain was obtained as an adh1 gene deletion strain 02T6Δadh strain.

  The PCR primers used in this example are shown in Table 1.

Example 2 Evaluation of alcohol dehydrogenase activity in mutant Rhizopus sp. The 02T6 strain and 02T6Δadh strain obtained in Example 1 were cultured, and the alcohol dehydrogenase (ADH) activity was measured.

(1) Culture of bacterial cells In a 500 mL Erlenmeyer flask with baffle (Asahi Glass), 200 mL seed medium (composition: 1% Difco Dextroze Broth (Becton Dickinson), 0.1% yeast extract B2 (Oriental Yeast Industry), 0.1% sodium glutamate, 0.002% tryptophan, 0.028% oleic acid, 0.5% sorbitan monolaurate (Leodol (registered trademark) SP-L10, manufactured by Kao Corporation), each having a concentration of% (w / v)), and a spore suspension of 02T6 strain or 02T6Δadh strain was inoculated so as to be 1 × 10 ³ -spore / mL-medium, and stirred at 27 ° C. for about 72 hours at 170 rpm. Next, the culture was filtered using a sterilized wire mesh having a mesh size of 250 μm, and the cells were collected on the filter. Inorganic culture solution 100mL (composition: 10% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate) , 0.009% zinc sulfate heptahydrate, 5.0% calcium carbonate, 0.002% tryptophan, 0.028% oleic acid, all in a concentration (% (w / v)) of 6.0-8 0.0 g was inoculated and cultured with stirring at 220 rpm at 27 ° C. for about 40 hours. The culture is filtered using a sterilized stainless steel screen filter holder (MILLIPORE), the cells are collected on the filter, and the cells are collected on the filter holder with about 50 mL of physiological saline. Washed twice. The physiological saline used for washing was removed by suction filtration. 40 mL of an inorganic culture solution (composition: 10% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate · 7) Hydrate, 0.009% zinc sulphate heptahydrate, 5.0% calcium carbonate, 0.002% tryptophan, 0.028% oleic acid, all in concentrations (% (w / v)) Shaking culture was performed at 35 ° C. and 170 rpm for 8 hours.

(2) Evaluation of alcohol dehydrogenase activity The cells were filtered from the culture obtained in (1) above using a sterilized stainless steel screen filter holder (MILLIPORE), and the cells were collected on the filter. Further, the cells were washed twice with about 50 mL of physiological saline on the filter holder. The physiological saline used for washing was removed by suction filtration. 0.3 g of the bacterial cells were collected in a 3 mL crushing tube (manufactured by Yasui Kikai Co., Ltd.), a 3 mL metal cone (manufactured by Yasui Kikai Co., Ltd.) was put in, the lid was closed, and then frozen with liquid nitrogen. The frozen 3 mL disruption tube was subjected to a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.), and the cells were disrupted at 1700 rpm for 10 seconds. Then, after suspending in 50 mM Tri-HCl (pH 7.5) solution supplemented with cComplete ULTRA Tables, Mini, EDTA-free, EASY pack (Roche), the mixture was centrifuged at 14500 rpm for 5 minutes, and the supernatant was disrupted. Liquid. The protein concentration of the microbial cell disruption solution was measured using a Quick Start Bradford Protein Assay (BIO-RAD).

The ADH activity of the cell disruption solution is described in Applied Biochemistry and Biotechnology, 2011, Vol. 164: 1305-1322, the following procedure was used. To 150 μL of the reaction solution (100 μL of 100 mM Tris-HCl (pH 7.5) solution and 50 μL of 700 μM NAD ⁺ solution), 10 μL of each appropriately lysed cell disruption solution was added and incubated at 35 ° C. for 5 minutes. Next, 40 μL of a 410 mM ethanol solution that had been preliminarily kept at 35 ° C. was added to start the enzyme reaction. 1 U of ADH activity was defined as the amount of enzyme that produced 1 μmol of NADH per minute at 35 ° C.

  The results are shown in Table 2. Compared with the 02T6 strain which is the parent strain, the ADH activity was reduced to about 6% in the 02T6Δadh strain.

Example 3 Evaluation of organic acid productivity in mutant Rhizopus genus 02T6 strain and 02T6Δadh strain were cultured in the same procedure as in Example 2 (1). However, the culture period was 2 days. The culture supernatant was sampled at 0, 4, 8, 10, 24, and 32 hours after the start of the culture, and glucose, fumaric acid, malic acid, succinic acid in the supernatant were sampled according to the procedure described in Reference Example 1 described later. The acid and ethanol were quantified, and then the selectivity of each substance was determined.

  Table 3 shows the selectivity of each substance at the time when complete consumption of glucose was confirmed (24T6 strain was 24 hours later and 02T6Δadh strain was 32 hours later). Compared with the parent strain 02T6, the 02T6Δadh strain had ethanol selectivity reduced to 91% and fumaric acid, malic acid and succinic acid selectivity improved to 106%, 161% and 108%, respectively.

Reference Example 1 Determination of glucose, fumaric acid, malic acid, succinic acid and ethanol in the culture <Analysis conditions>
Glucose, glucose, fumaric acid, malic acid, succinic acid, and ethanol in the culture supernatant were quantified using an HPLC apparatus LaChrom Elite (manufactured by Hitachi High-Technologies). As the analytical column, a guard column ICS Sep ION-300 Guard Column Cartridge (4.0 mm ID × 20 mm, manufactured by Transgenomic) was connected to an organic acid analysis polymer column ICS Sep ICE-ION-300 (7.8 mm I.D.). D. × 300 mm, manufactured by Transgenomic) was used for elution under the conditions of an eluent 0.01N sulfuric acid, a flow rate of 0.5 mL / min, and a column temperature of 50 ° C. A suggested refractive index detector (RI detector) was used to detect glucose and ethanol, and a UV detector (detection wavelength 210 nm) was used to detect fumaric acid, malic acid and succinic acid. A culture supernatant sample to be subjected to HPLC analysis was previously diluted 3-fold with 0.86 M sodium sulfate solution and then appropriately diluted with 37 mM sulfuric acid, and then AcroPrep 96-well Filter Plates (0.2 μm GHP membrane, manufactured by Pall). Was used to remove insolubles.

<Calculation of selectivity>
The selectivity of a substance is the actual value obtained from the culture relative to the maximum equivalent of the substance that can be theoretically obtained from the culture (calculated from the glucose consumption of the culture). Refers to the ratio of equivalents of substances. That is, the selectivity is obtained by the following formula.
Selectivity = product equivalent / theoretical maximum amount (where theoretical maximum amount = equivalent glucose consumption × [correction coefficient])
The theoretical maximum amount and correction factor for the substance to be measured are as follows:
[Theoretical maximum amount] [Correction factor]
Ethanol: 1 mol of glucose up to 2 mol 1/2
Fumaric acid: 2 mol of glucose up to 3 mol 2/3
Malic acid: 2 mol of glucose up to 3 mol 2/3
Succinic acid: 2 mol of glucose up to 3 mol 2/3

Claims (20)

  A mutant Rhizopus genus having reduced activity of alcohol dehydrogenase.   The mutant Rhizopus bacterium according to claim 1, wherein the adh gene is deleted or inactivated. The adh gene is:
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
A polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity; and
A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity;
The mutant Rhizopus bacterium according to claim 2, which is at least one selected from the group consisting of:   The mutant Rhizopus bacterium according to any one of claims 1 to 3, wherein the activity of alcohol dehydrogenase is reduced to 50% or less compared to that before mutation.   The mutant Rhizopus bacterium according to any one of claims 1 to 4, wherein the Rhizopus genus is Rhizopus delmar or Rhizopus oryzae.   The mutant Rhizopus bacterium according to any one of claims 1 to 5, wherein the organic acid productivity is improved.   A method for producing a mutant Rhizopus, comprising reducing the activity of alcohol dehydrogenase in Rhizopus.   A method for improving organic acid productivity of Rhizopus sp., Comprising reducing the activity of alcohol dehydrogenase in Rhizopus sp.   The method according to claim 7 or 8, comprising deleting or inactivating the adh gene in the genus Rhizopus. The adh gene is:
A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
A polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence represented by SEQ ID NO: 1 and encoding a polypeptide having alcohol dehydrogenase activity;
A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity; and
A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 and having alcohol dehydrogenase activity;
The method of claim 9, wherein the method is at least one selected from the group consisting of:   The method according to claim 9 or 10, wherein the deletion or inactivation of the adh gene is performed by genome editing of the adh gene locus using an artificial DNA cleaving enzyme.   12. The method of claim 11, wherein the genome editing is performed by TALEN, Crispr-cas9 system, or ZFN.   The method according to claim 12, wherein the genome editing is introducing a TALEN peptide or a polynucleotide encoding the same into the Rhizopus bacterium. The method according to claim 12 or 13, wherein the TALEN comprises the following polypeptides (L) and (R):
Polypeptide (L):
Has a TAL effector with 17 repeats linked;
Among the 17 repeats, the 1st to 16th repeats from the upstream are each an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 11, or the amino acid sequence represented by SEQ ID NO: 11 Consisting of an amino acid sequence in which no more than 5 amino acid residues are deleted, inserted, substituted or added to
The 17th repeat from the upstream of the 17 repeats consists of an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 27;
The TAL effector recognizes the sequence represented by SEQ ID NO: 9.
Polypeptide (R):
Has a TAL effector with 16 repeats linked;
Among the 16 repeats, the 1st to 15th repeats from the upstream are each an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 28, or the amino acid sequence represented by SEQ ID NO: 28 Consisting of an amino acid sequence in which no more than 5 amino acid residues are deleted, inserted, substituted or added to
The 16th repeat from the upstream of the 16 repeats consists of an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 43;
The TAL effector recognizes the sequence represented by SEQ ID NO: 10. The method according to claim 12 or 13, wherein the polynucleotide encoding TALEN is the following i) and ii):
i) A nucleotide sequence represented by SEQ ID NO: 3, or a polynucleotide comprising a nucleotide sequence having at least 95% identity thereto, or an amino acid sequence represented by SEQ ID NO: 4, or at least 95% identity thereto A polynucleotide encoding a polypeptide comprising an amino acid sequence, and
ii) a nucleotide sequence represented by SEQ ID NO: 5 or a polynucleotide comprising a nucleotide sequence having at least 95% identity thereto, or an amino acid sequence represented by SEQ ID NO: 6, or at least 95% identity thereto Polynucleotide encoding polypeptide comprising amino acid sequence   The method according to any one of claims 13 to 15, further comprising introducing an exonuclease or a polynucleotide encoding the same into the Rhizopus bacterium. A polynucleotide encoding the exonuclease,
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 7 and encoding a polypeptide having exonuclease activity;
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 69;
A polynucleotide comprising a nucleotide sequence having at least 95% identity to the nucleotide sequence represented by SEQ ID NO: 69 and encoding a polypeptide having exonuclease activity;
A polynucleotide encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8; or a polypeptide comprising an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 8 and having exonuclease activity The encoding polynucleotide,
The method of claim 16, wherein   The method according to any one of claims 7 to 17, wherein the Rhizopus genus is Rhizopus delmar or Rhizopus oryzae.   A method for producing an organic acid, comprising culturing the mutant Rhizopus bacterium according to any one of claims 1 to 6.   The method for producing an organic acid according to claim 19, wherein the organic acid is fumaric acid, lactic acid, succinic acid, malic acid, or α-ketoglutaric acid.