Method for producing RNA

Patent metadata

Field	Value
Publication number	US-11046986-B2
Application number	US-201916582385-A
Country	US
Kind code	B2
Filing date	Sep 25, 2019
Priority date	Mar 28, 2017
Publication date	Jun 29, 2021
Grant date	Jun 29, 2021

Abstract

A method for producing RNA is provided. Objective RNA is produced by culturing a coryneform bacterium having an expression unit for the objective RNA, which has been modified so that the activity of ribonuclease III is reduced, in a medium, to express the objective RNA and accumulate the objective RNA in cells of the bacterium, and collecting the objective RNA from the cells.

First claim

We claim:

1. A method for producing objective RNA, the method comprising:
A) culturing a coryneform bacterium having an expression unit for the objective RNA in a medium, to express the objective RNA and accumulate the objective RNA in cells of the bacterium; and
B) collecting the objective RNA from the cells,
wherein the bacterium has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain by deletion of a gene encoding ribonuclease III;
wherein the ribonuclease III is a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 52;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 52, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having ribonuclease III activity; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 52, and having ribonuclease III activity.

2. The method according to claim 1 , wherein the bacterium comprises 5 copies/cell or more of the expression unit.

3. The method according to claim 1 , wherein the bacterium comprises 70 copies/cell or more of the expression unit.

4. The method according to claim 1 , wherein the bacterium comprises a vector containing the expression unit.

5. The method according to claim 1 , wherein the expression unit comprises a promoter sequence that functions in the coryneform bacterium and a nucleotide sequence encoding the objective RNA in the direction from 5′ to 3′.

6. The method according to claim 5 , wherein the promoter sequence is a promoter derived from a phage.

7. The method according to claim 5 , wherein the promoter sequence is F1 promoter or T7 promoter.

8. The method according to claim 5 , wherein the promoter sequence is a promoter selected from the group consisting of:
(a) a promoter comprising the nucleotide sequence of SEQ ID NO: 13 or 78; and
(b) a promoter comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 13 or 78.

9. The method according to claim 1 , wherein the bacterium is a bacterium belonging to the genus Corynebacterium.

10. The method according to claim 1 , wherein the bacterium is Corynebacterium glutamicum.

Assignees

• Ajinomoto Kk

Inventors

• Hashiro Shuhei
• Yasueda Hisashi
• Mitsuhashi Mayu
• Mashko Sergei Vladimirovich
• Krylov Aleksandr Aleksandrovich
• Lobanova Yuliya Sergeevna

Classifications

• C12R2001/28

  Micrococcus glutamicus {; Corynebacterium glutamicum} · CPC title

• C12N15/77 Primary

  for Corynebacterium; for Brevibacterium · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12R2001/15

  Corynebacterium · CPC title

• C12R2001/13

  Brevibacterium · CPC title