pheS ^* , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens

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• 作者：Chaoyang Zhou ; Lingling Shi ; Bin Ye ; Haichao Feng&#8230
• 关键词：B. amyloliquefaciens ; Counter ; selectable marker ; pheS* ; Unmarked genetic manipulation
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：101
• 期：1
• 页码：217-227
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Microbiology; Microbial Genetics and Genomics; Biotechnology;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-0614
• 卷排序：101

文摘

Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS* was constructed through a point mutation of the gene pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5 mM p-chloro-phenylalanine, 100 % of B. amyloliquefaciens strain SQR9 cells carrying pheS* were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS* and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS* as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS* in other Bacillus species was preliminarily assessed. The expression of PheS* in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS* could be used as a counter-selectable marker (CSM) in these strains.