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Objective Bacillomycin L, a cyclic lipopeptide antibiotic produced by Bacillus velezensis Bs916, has been demonstrated to possess strong antifungal activity. However, its low yield has become a critical bottleneck limiting its large-scale application. This study aims to identify the negative transcriptional regulators involved in bacillomycin L biosynthesis and, based on this, adopt a dual strategy combining genetic engineering and fermentation process optimization to promote its large-scale production. Methods Homologous recombination was employed to construct single (ΔresD, ΔabrB) and double (ΔresDΔabrB) knockout strains. The regulatory characteristics were elucidated by HPLC, inhibition zone measurement, and RT-qPCR. Furthermore, EMSA and DNase I footprinting assays were conducted to investigate the binding activity and identify the specific binding sites of these transcription factors with the flanking sequences of the transcription initiation site of the Bac gene cluster. Finally, fermentation process optimization was performed in a bioreactor to further enhance the production of bacillomycin L. Results HPLC results demonstrated that the bacillomycin L yields of all the mutant strains cultured in the LB media significantly increased by 5.8, 11.3, and 12.0 folds compared with that of the wild-type strain. The antagonistic activities of the mutants against plant pathogenic fungi exhibited corresponding increases. RT-qPCR results further confirmed that both ResD and AbrB acted as negative regulators of bacillomycin L biosynthesis. EMSA experiments revealed that both ResD and AbrB possessed strong binding activities with their target sequences. DNase I footprinting assays further elucidated that AbrB exhibited a propensity for binding to A+T-rich gene fragments and displayed extensive DNA-binding capabilities, directly interacting with the promoter region, 5′ UTR, and coding regions of the Bac gene cluster. Unfortunately, the specific binding site of ResD remained to be identified. Under fermentation conditions, the genetically engineered strain ΔresDΔabrB achieved gram-per-liter level production of bacillomycin L in an optimized glucose-mineral salts medium. This result greatly promoted the large-scale production of bacillomycin L. Conclusion Adopting a dual strategy that combines genetic engineering with fermentation process optimization effectively breaks the bottleneck of low yields for Bacillus-derived antimicrobial peptides. This study provides a reference for the mass production of other microbial secondary metabolites.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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