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

Zhou, Chaoyang, Shi, Lingling, Ye, Bin, Feng, Haichao, Zhang, Ji, Zhang, Ruifu, Yan, Xin
Applied microbiology and biotechnology 2017 v.101 no.1 pp. 217-227
Bacillus amyloliquefaciens, Bacillus cereus, Bacillus subtilis, chromosome elimination, genes, genetic engineering, hosts, metabolic engineering, metabolites, phenylalanine-tRNA ligase, plant growth, plant growth-promoting rhizobacteria, plant pathogens, point mutation
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.