Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering.

Applied Microbiology and Biotechnology
Meng WuGuangdong Shang

Abstract

Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C. glutamicum. Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C. glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C. glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five ...Continue Reading

References

Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Jun 7, 2001·Proceedings of the National Academy of Sciences of the United States of America·H M EllisD L Court
Apr 5, 2002·Genome Research·Vitaliy KolisnychenkoGyörgy Pósfai
Apr 19, 2005·Applied Microbiology and Biotechnology·Nobuaki SuzukiHideaki Yukawa
Apr 22, 2005·Applied Microbiology and Biotechnology·Nobuaki SuzukiHideaki Yukawa
Apr 7, 2006·Applied and Environmental Microbiology·Alexandra E Bloor, Rocky M Cranenburgh
Mar 18, 2008·Environmental Microbiology·Ronald S FlannaganMiguel A Valvano
Mar 3, 2011·Journal of Microbiological Methods·Naoko OkibeHideaki Yukawa
Jul 31, 2014·Nucleic Acids Research·Jee-Hwan Oh, Jan-Peter van Pijkeren
Aug 15, 2015·Applied Microbiology and Biotechnology·Jean Paul OuedraogoArthur F J Ram
Feb 2, 2016·ACS Synthetic Biology·Sara CletoTimothy K Lu
May 26, 2016·EcoSal Plus·Kenan C Murphy
Oct 22, 2016·FEMS Microbiology Letters·Zhongqiu ChenGuangdong Shang
May 5, 2017·Nature Communications·Yu JiangSheng Yang
May 30, 2017·Bioresource Technology·Jin-Ho Lee, Volker F Wendisch
Jun 27, 2017·Metabolic Engineering·Jae Sung ChoSang Yup Lee
Nov 15, 2018·Frontiers in Microbiology·Fernando Pérez-GarcíaVolker F Wendisch
Nov 28, 2018·Applied and Environmental Microbiology·Elena-Stella TheophilouGarry W Blakely
Dec 12, 2018·Current Opinion in Biotechnology·Jae Woong ChoiKi Jun Jeong
Feb 15, 2019·Applied Microbiology and Biotechnology·Zhixin WuGuangdong Shang
Mar 18, 2019·Applied Microbiology and Biotechnology·Jing LiGuangdong Shang

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Citations

Apr 27, 2021·Frontiers in Microbiology·Qingzhuo WangSheng Yang

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Methods Mentioned

BETA
gene knock-in
PCR
electrophoresis
PCRs

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