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

文献类型： 外文期刊

第一作者： Yang, Ying-Jie

作者： Yang, Ying-Jie;Singh, Raghvendra Pratap;Li, Yue-Zhong;Sheng, Duo-Hong;Yang, Ying-Jie;Zhang, Cheng-Sheng;Li, Yi-Qiang;Singh, Raghvendra Pratap;Lan, Xin

作者机构：

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

期刊名称：BIOMOLECULES （ 影响因子：4.879； 五年影响因子：5.362 ）

ISSN： 2218-273X

年卷期： 2018 年 8 卷 4 期

页码：

收录情况： SCI

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.

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