Construction Of Iturin A Hyper-producers And Optimization On The Fermentation Conditions In Bacillus Amyloliquefaciens

As a widely studied lipopeptide produced by Bacillus spp,iturin A is an ideal biological control agent with the aim of reducing the use of chemical pesticides in agriculture.At present,the production of iturin A on a commercial scale has not been realized because of low yields and high production costs.Following homologous recombination,iturin A mutants of Bacillus amyloliquefaciens HZ12-Pbac Aitu,HZ12-P43itu and HZ12-Psrfitu were constructed with the bacitracin operon promoter(P_{bac A}),constitutive promoter（P43）,and the surfactin operon promoter(P_srf),respectively.The mutants HZ12-Pbac Aitu,HZ12-P43itu produced950.08 mg/L and 735.12 mg/L of iturin A,respectively,almost 1.32 times and 0.80 higher than the iturin A produced from the wild strain HZ12（408.97 mg/L）while the iturin A production of HZ-12-Psrfitu was 51.15 mg/L,which was significantly lower than that of the original strain HZ-12.A consistent change trend was observed itu D gene transcription.It further demonstrated that the promoters with different strengths regulated genes expression of B.amyloliquefaciensat the transcriptional level,and thus affected the iturin A expression in this strain.In this study,we constructed the completed iturin A high yield engineering strain HZ12-Pbac Aitu,and used the method of single factor experiment and orthogonal design to optimize the fermentation medium and condition of iturin A shake flask fermentation.The optimum carbon source for the synthesis of iturin A was corn starch,the organic nitrogen source was more suitable for the synthesis of iturin A and the soybean meal was the optimum nitrogen source.The concentration of inorganic salts KH₂PO₄ and Fe SO₄·7H₂O also significantly affected the production of iturin A while Mg SO₄·7H₂O and Mn SO₄·H₂O have no significant effect on the production of iturin A.The optimized medium composition was as follows:50 g/L corn starch,70 g/L soybean meal,1 g/L KH₂PO₄,0.50 g/L Mg SO₄·7H₂O,0.30 g/L.And the fermentation conditions were as follows:when the culture temperature was 28°C,the seed liquid was 9 h,the inoculation amount was（v/v）2%,the initial p H of the fermentation was 6.0.Under the optimal culture medium and culture conditions,HZ-12-Pbac Aitu produced the highest iturin A concentration of 1976.24 mg/L,which was 1.08 times higher than that of the starting medium,and increased by 3.83 times compared with that of the wild type strain HZ-12.In order to solve the serious foam problem in the fermentation process of antimicrobial lipopeptide,We attempts to knock out srf AC,the synthetic genes of surfactin,to obtain the deletion strain HZ12-Pbac Aitu-Δsrf AC.Compared with its parent strain HZ12-Pbac Aitu,the mutant HZ12-Pbac Aitu-Δsrf AC produces less foam and the use of antifoam was reduced by 20.The yield of iturin A by Bacillus amyloliquefaciens HZ12-Pbac Aitu-Δsrf AC in the 5L fermentor reached 1400 mg/L,which meets current industrial production needs.