Abstract
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.
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Acknowledgments
The authors would like to acknowledge Dr. Natalia P. Zakataeva from the Ajinomoto-Genetika Research Institute for providing the plasmid pNZT1.
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The authors would like to express their thanks for financial support from the 863 Plan (2014AA020543), the 973 Plan (2015CB150505) and the National Natural Science Foundation (31300099 and 31470225) of China.
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Zhou, C., Shi, L., Ye, B. et al. pheS *, an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens . Appl Microbiol Biotechnol 101, 217–227 (2017). https://doi.org/10.1007/s00253-016-7906-9
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DOI: https://doi.org/10.1007/s00253-016-7906-9