Compound Mutagenesis to Improve the Antibacterial Ability of Surfactin from Bacillus subtilis
LI Guang-Yue1, HU Wen-Feng1,2, LI Xue-Ling1*
1 College of Food Science, South China Agricultural University, Guangzhou 510642, China; 2 Bioforte Biotechnology (Shenzhen) Co., Ltd., Shenzhen 518118, China
Abstract：Compared with chemical surfactants, surfactin has the characteristics of low toxicity, environmental friendliness and reduced surface tension, which has great application prospects in agriculture, food, medicine, cosmetics and other fields, but there are problems of low yield and high cost in production. Multiple mutagenesis methods (physical, chemical and aerospace mutagenesis) were used to select and mutate Bacillus subtilis, and a genetically stable compound mutagenic strain FHYB201030 was obtained by combining blood plate, cetylpyridinium chloride-bromothymol blue (CPC-BTB) colorimetric method and high performance liquid chromatography screening through mutagenic selection in this study. Whole genome sequencing was performed on the starting strain (YUAN. 0) and the mutant strain (FUYB201030), respectively. The fermentation broth of the mutant strain (FUYB201030) was collected and purified, and then surfactin obtained was assayed for yield and in vitro antibacterial activity. The results showed that the surfactin production of FHYB201030 was 16.8 times higher than that of YUAN. 0, and the production was up to 353 mg/L. The whole genome of YUAN.0 was composed of a 5 209 013 bp complete circular chromosome and a 299 348 bp complete circular plasmid. Through preliminary analysis of the resequencing data of FHYB201030, it was found that a total of 450 SNP mutation sites in CDS of the strain were caused by complex mutagenesis, which mainly involved 28 gene sequences. Combined mutagenesis not only increased the yield of Bacillus subtilis, but also affected the growth cycle of Bacillus subtilis and improved the antibacterial ability of surfactin. Compared with YUAN. 0, the same concentration of the surfactin (40 mg/L) synthesized by FHYB201030 showed enhanced inhibitory effect on bacteria and fungi, and had significant inhibitory effect on the growth of indicator bacteria. In conclusion, the combined mutagenesis method could effectively improve the ability of Bacillus subtilis to produce surfactin and improve the activity of surfactin, which provides a basis for further research and application of surfactin.
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