Mutant of genus Rhizopus

Patent metadata

Field	Value
Publication number	US-10947522-B2
Application number	US-201716074162-A
Country	US
Kind code	B2
Filing date	Feb 1, 2017
Priority date	Feb 4, 2016
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

Abstract

The present invention provides a fungus of the genus Rhizopus having high productivity of an organic acid. The present invention also provides a mutant of the genus Rhizopus with reduced pyruvate decarboxylase activity.

First claim

What is claimed is:

1. A method for improving organic acid productivity of a Rhizopus fungus, comprising:
deleting or inactivating a pdc gene in the Rhizopus fungus, wherein
the pdc gene is at least one polynucleotide selected from the group consisting of:
a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1;
a polynucleotide consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide which has pyruvate decarboxylase activity;
a polynucleotide comprising a nucleotide sequence that has 1-20 deletions, insertions, substitutions or additions of nucleotides as compared to the nucleotide sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide which has pyruvate decarboxylase activity;
a polynucleotide encoding a polypeptide which consists of the amino acid sequence set forth in SEQ ID NO: 2;
a polynucleotide encoding a polypeptide which consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that has pyruvate decarboxylase activity; and
a polynucleotide encoding a polypeptide which comprises an amino acid sequence that has 1-10 deletions, insertions, substitutions or additions of amino acid residues as compared to the amino acid sequence set forth in SEQ ID NO: 2 and that has pyruvate decarboxylase activity;

and culturing the fungus to produce the organic acid,
wherein the organic acid is selected from the group consisting of fumaric acid, lactic acid, succinic acid, malic acid, and α-ketoglutaric acid,
and wherein production of the organic acid is improved in the Rhizopus fungus compared to the production of the organic acid before deletion or inactivation of the pdc gene.

2. The method of claim 1 , wherein the Rhizopus is Rhizopus oryzae or Rhizopus delemar.

3. The method of claim 1 , further comprising collecting the organic acid from the culture after the culturing.

4. The method of claim 1 , wherein the organic acid is fumaric acid, succinic acid or malic acid.

5. The method of claim 1 , wherein the culturing is aerobic culturing.

6. The method of claim 1 , wherein the deleting or inactivating of the pdc gene is performed by genome editing of a pdc gene locus using a programmable nuclease.

7. The method of claim 6 , wherein the genome editing is transfer of TALEN peptides or polynucleotides encoding the TALEN peptides to the Rhizopus fungus.

8. The method of claim 7 , wherein the TALEN peptides consist of the following polypeptides (L) and (R):
the polypeptide (L) being
a polypeptide which consists of the amino acid sequence set forth in SEQ ID NO: 4;
a polypeptide which consists of an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 4 and has a TAL effector targeting the sequence set forth in SEQ ID NO: 9 and a DNA cleavage domain consisting of Fok1-like DNA nuclease;
a polypeptide which is encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3; or
a polypeptide which is encoded by a polynucleotide consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence set forth in SEQ ID NO: 3 and has a TAL effector targeting the sequence set forth in SEQ ID NO: 9 and a DNA cleavage domain consisting of Fok1-like DNA nuclease, and

the polypeptide (R) being
a polypeptide which consists of the amino acid sequence set forth in SEQ ID NO: 6;
a polypeptide which consists of an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 6 and has a TAL effector targeting the sequence set forth in SEQ ID NO: 10 and a DNA cleavage domain consisting of Fok1-like DNA nuclease;
a polypeptide which is encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 5; or
a polypeptide which is encoded by a polynucleotide consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence set forth in SEQ ID NO: 5 and has a TAL effector targeting the sequence set forth in SEQ ID NO: 10 and a DNA cleavage domain consisting of Fok1-like DNA nuclease.

9. The method of claim 7 , further comprising transferring an exonuclease or a polynucleotide encoding the exonuclease to the Rhizopus fungus.

Assignees

• Kao Corp

Inventors

• Tsuboi Yuichi

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12R2001/845

  Rhizopus · CPC title

• C12N1/145

  Fungi isolates · CPC title

• C12P7/50

  having keto groups, e.g. 2-ketoglutaric acid · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title