Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System

Biomolecules

卷:8

Xin Lan¹  Cheng-Sheng Zhang²  Yi-Qiang Li²  Ying-Jie Yang³  Yue-Zhong Li³  RaghvendraPratap Singh³  Duo-Hong Sheng³ 

[1] Department of Bio-Chemistry, Qingdao Technical College, Qingdao 266555, China;

[2] Marine Agriculture Research Center, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China;

[3] State Key Laboratory of Microbial Technology, Microbiology Technology Institute, Shandong University, Qingdao 266237, China;

关键词: Cre/loxP;    large fragment deletion;    Myxococcus xanthus;    loxP spacer mutant;    copper inducible promoter;    Escherichia coli-Myxococcus shuttle plasmid;    genome editing;   

DOI  :  10.3390/biom8040137

来源: DOAJ

【 摘 要 】

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter P_cuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter P_cuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

【 授权许可】

Unknown