Bacillus Amyloliquefaciens And Its Biosynthesis Conditions Of Cyclic Lipopeptides

Bacillus amyloliquefaciens is a member of Bacillus genus which is often considered to be microbial factory for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth. Among these active compounds, the most widely studied chemical is cyclic lipopeptides (CLPs). However, due to the low capacity of lipopeptide production, CLPs produced by Bacillus has not been industrialized so far. In order to provide basic foundation and application of CLPs and its producing bacteria, in this study, we focus on the biocontrol activities and the conditions for CLPs biosynthesis of an endophytic strain CC09that was isolated from Cinnamomum camphora, through strain identification, active compound isolation and structure elucidation, fermentation optimization, and RNA polymerase mutations that may lead to high-yield CLPs. The major results are as follows:1. The strain CC09was identified to be Bacillus amyloliquefaciens based on morphology, physio-biochemical reaction and16s rDNA/rpoB sequence analysis. The inhibition rate of CC0948h culture filtrate against phytopathogen Glomerella glycines, Rizoctonia solani, Alternaria alternate, Fusarium graminearum, Fusarium oxysporum and Phytophthora capsici were95%、86%、81%、57%、37%and18%respectively.2. Macroporous resin, silicone gel and Sephadex LH-20were used to isolate the antifungal compounds from CC09culture filtrate. Three active chemicals from the filtrate were identified as Iturin A2, A3and A6by extensive spectral analysis. Through the Polymerase Chain Reaction by specific primers and analysis of the PCR products sequences, not only systhetase coding gene of Iturin A but also Surfactin and Fengycin can be amplified from CC09genome.3. The optimized culture condition for CC09strain is as follows:Culture medium (1L):5g soluble starch,15g peptone and yeast extract (m:m=3:1), lgNaCl.Inoculation:1%(v/v) of the overnight cultures of CC09strain in a250mL flask containing50mL culture medium, and incubation in a shaking incubator at120rpm,28℃for36.5h. The Iturin A production is690μg/mL which is4times higher than that before optimization.In addition, the biological safety evaluation of CC09and its fermentation broth towards silkworm is also carried out.4. In order to improve the strain’s capacity for production of the antifungal metabolites,77rif mutants had been isolated from the strain CC09and30%of them have improved the capacity of producing antifungal metabolites.13mutants had been sequenced for rpoB gene coding for β subunit of RNAP and their effects on antifungal activity against phytopathogen F. graminearum in vitro were evaluated. Six types of mutations, including H485R, H485Y, H485C, Q472R, S490L and S490L/S617F can be found. Among them, H485R mutants enhance the production of Iturin A and surfactin by2-3fold as high as that of the wild strain. H485C mutants don’t influence the antibiotic production. Other mutants significantly decrease the biosynthesis of those CLPs. These mutants with different antibiotic production may offer a good system to elucidate the regulation of CLPs transcription.In summary, the ability of the Bacillus amyloliquefaciens CC09to produce the active metabolite CLPs can be improved by the fermentation conditions optimization and the RNA polymerase mutations. This lay a foundation for CLPs’ industrial application and the CLPs transcriptional regulation mechanism research.