Title:
METHOD FOR PREPARING CORYNEBACTERIUM MUTANT STRAIN, USING CRISPR/CAS SYSTEM, RECOMBINASE, AND SINGLE-STRANDED OLIGODEOXYRIBONUCLEIC ACID
Document Type and Number:
WIPO Patent Application WO/2018/182361
Kind Code:
A1
Abstract:
The present invention relates to a method for preparing a bacteria mutant strain, comprising the steps of: (a) primarily transforming a normal bacteria into an enzyme-expressing vector which has temperature sensitivity or antibiotic sensitivity and expresses (i) a recombinase; (b) making the primarily transformed bacteria obtained in step (a) to be a competent cell; (c) secondarily transforming the competent cell obtained in step (b) by introducing (i) a single-stranded oligodeoxyribonucleic acid (ssODN) which binds complementarily to the target gene and (ii) a first vector which expresses a guide (RNA) and has antibiotic sensitivity or temperature sensitivity; and (e) removing the inserted enzyme-expressing vector and the first vector from the secondarily transformed bacteria, wherein at least one of the enzyme-expressing vector and the first vector expresses a Cas protein. A method according to the present invention can delete a target gene effectively, select a recombinant microorganism rapidly and at high efficiency, and remove all foreign vectors from the finally selected recombinant microorganism, finding very useful applications in the industrial use of recombinant Corynebacterium.
Inventors:
LEE SANG YUP (KR)
CHO JAE SUNG (KR)
CHOI KYEONG ROK (KR)
CHO JAE SUNG (KR)
CHOI KYEONG ROK (KR)
Application Number:
PCT/KR2018/003784
Publication Date:
October 04, 2018
Filing Date:
March 30, 2018
Export Citation:
Assignee:
KOREA ADVANCED INST SCI & TECH (KR)
International Classes:
C12N15/77; C12N15/113; C12N15/64; C12N15/65; C12P13/00
Domestic Patent References:
| WO2016205703A1 | 2016-12-22 |
Foreign References:
| KR20120046522A | 2012-05-10 | |||
| US6303383B1 | 2001-10-16 |
Other References:
JIANG, Y. ET AL.: "Multigene Editing in the Escherichia Coli Genome via the CRISPR-Cas9 System", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 81, no. 7, 2015, pages 2506 - 2514, XP055444637
NAKAMURA, J. ET AL.: "Mutations of the Corynebacterium Glutamicum NCgl1221 Gene , Encoding a Mechanosensitive Channel Homolog, Induce L-Glutamic Acid Production", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 73, no. 14, 18 May 2007 (2007-05-18), pages 4491 - 4498, XP002691368
RAN, F. A. ET AL.: "Genome Engineering Using the CRISPR-C'as9 System", NATURE PROTOCOLS, vol. 8, no. 11, 2013, pages 2281 - 2308, XP009174668
BINDER, S. ET AL.: "Recombineering in Corynebacterium Glutamicum Combined with Optical Nanosensors: a General Strategy for Fast Producer Strain Generation", NUCLEIC ACIDS RESEARCH, vol. 41, no. 12, 2013, pages 6360 - 6369, XP055111472
CHO, J. S. ET AL.: "CRISPR/Cas9-coupled Recombineering for Metabolic Engineering of Corynebacterium Glutamicum", METABOLIC ENGINEERING, vol. 42, 23 June 2017 (2017-06-23), pages 157 - 167, XP085136204
NAKAMURA, J. ET AL.: "Mutations of the Corynebacterium Glutamicum NCgl1221 Gene , Encoding a Mechanosensitive Channel Homolog, Induce L-Glutamic Acid Production", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 73, no. 14, 18 May 2007 (2007-05-18), pages 4491 - 4498, XP002691368
RAN, F. A. ET AL.: "Genome Engineering Using the CRISPR-C'as9 System", NATURE PROTOCOLS, vol. 8, no. 11, 2013, pages 2281 - 2308, XP009174668
BINDER, S. ET AL.: "Recombineering in Corynebacterium Glutamicum Combined with Optical Nanosensors: a General Strategy for Fast Producer Strain Generation", NUCLEIC ACIDS RESEARCH, vol. 41, no. 12, 2013, pages 6360 - 6369, XP055111472
CHO, J. S. ET AL.: "CRISPR/Cas9-coupled Recombineering for Metabolic Engineering of Corynebacterium Glutamicum", METABOLIC ENGINEERING, vol. 42, 23 June 2017 (2017-06-23), pages 157 - 167, XP085136204
Attorney, Agent or Firm:
LEE, Cheo Young et al. (KR)
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