Metabolic Mechanism Of Highly-efficient Biosynthesis Of Surfactin And Its Function

Natural Bacillus isolates generate limited amounts of surfactin?<10%of their biomass?,which functions as an antibiotic or signalling molecule in inter-/intra-specific interactions.Surfactin is also a powerful biosurfactant with significant biological activities of antibacteria,antiviral,antitumor,and antithrombotic,which make it possess broad application prospects in various industrial fields,including oil recovery,biopesticides,food processing,cosmetic,and pharmaceuticals.However,until now it is still difficult to realize the industrial preparation and wide application of surfactin.The main reasons can be concluded as below:1)the surfactin yields of most natural Bacillus isolates are very low,2)the key factors that limit the high efficiency of synthesis are still unclear,and 3)the cost including production,separation,and purification of surfactin are generally very high.In this study,we firstly set up a high-throughput screening strategy for high surfactin yield strains screening;then we used omics'biotechnology to systemically unravel the highly efficient biosynthesis of surfactin in B.amyloliquefaciens MT45,and subsequently used metabolic engineering technology to artificially design the key pathway to address the bottlenecks of highly efficient production of surfactin.In addition,we studied the natural function of surfactin high-producing strain in the Chinese liquor fermentation process,and the processes that waste distillers'grains were directly used to economically produce surfactin by decreasing the production costs.The main contents are as follows:?1?Obtaining natural high-yield strains is the first and most important step to achieve the goal of industrial surfactin production.In this study,a strain of Bacillus amyloliquefaciens,which produced 3.37 g?L^-1 surfactin?³0-fold of the average yield?,was isolated from the special natural environment?high temperature Daqu?by high-throughput screening strategy combined with blood agar plate and specific PCR screening.The single factor experiment and the CCD experiment were used to optimize the fermentation conditions.Surfactin production was significantly improved more than2 times,reaching to 7.9 g?L^-1,which was the highest yield of wild Bacillus strains.?2?The natural function of surfactin in Daqu was revealed by studying the microbial interactions between B.amyloliquefaciens and other brewing microorganisms.Streptomyces spp.,producing geosmin,have been regarded as the most frequent and serious microbial contamination causing earthy off-flavor in Chinese liquor.It is therefore necessary to control the Streptomyces community during liquor fermentation.Biological control,using the native microbiota present in liquor making,appears to be a better solution than chemical methods.The objective of this study was to isolate native microbiota antagonistic toward Streptomyces spp.and then to evaluate the possible action mode of the antagonists.Fourteen Bacillus strains,isolated from different Daqu?the fermentation starter?,showed antagonistic activity against Streptomyces sampsonii,which belongs to the dominant geosmin producer.Among them,Bacillus subtilis 2-16 and B.amyloliquefaciens 1-45 from Maotai Daqu could significantly inhibit the growth of S.sampsonii by 57.8%and 84.3%respectively,and thoroughly prevent the geosmin production in the simulated fermentation experiments?inoculation ratio 1:1?.To probe the biocontrol mode,the ability of strain 2-16 and 1-45 to produce antimicrobial metabolites and to reduce geosmin from fermentation system was investigated.Antimicrobial substances were identified as lipopeptides by ultra-performance liquid chromatography tandem electrospray ionization/quadrupole-time-of-flight mass spectrometry?UPLC-ESI/Q-TOF MS?and in vitro antibiotic assay.In addition,strains 2-16 and 1-45 could remove 45%and 15%of the geosmin respectively in the simulated solid-state fermentation.The results highlighted the potential of biocontrol,and using native Bacillis species in Daqu could eco-friendly prevent the Streptomyces spp.and geosmin contamination in Chinese liquor fermentation.?3?Bacillus in Daqu can synergetically use DGS as carbon source to produce surfactin.B.amyloliquefaciens MT45 could grow with DGS as sole carbon source to produce 1.04 g?L^-1 surfactin.However,the low amylase activity of MT45 limited the sugar supply and subsequent surfactin production.Therefore,MT45 was co-cultured with Bacillus strains that exhibited remarkable hydrolases activities.Surfactin yield increased by 50%when MT45 was co-cultured with B.amyloliquefaciens X82 that showed no product inhibition effect and did not develop extracellular matrix.The inoculation ratio of X82 greatly influenced the sugar supply,cellular growth,and surfactin production of the co-culture fermentation.The maximum surfactin titration(3.4 g?L^-1)was obtained when MT45 and X82 was co-cultured with inoculation ratio at1:0.5,using 200 g?L^-1 DGS.This work highlights the feasiblity of using industrial waste DGS as promising feedstocks to produce value-added surfactin by co-culture fermentation.?4?In order to systemically unravel the efficient surfactin biosynthetic process of B.amyloliquefaciens MT45,the genome and transcriptome of this bacterium were compared with those of B.amyloliquefaciens type strain DSM7^T.MT45 possesses a smaller genome while containing more unique transporters and resistance-associated genes.Comparative transcriptome analysis revealed notable enrichment of the surfactin synthesis pathway in MT45,including central carbon metabolism and fatty acid biosynthesis to provide sufficient quantities of building precursors.Most importantly,the modular surfactin synthase overexpressed?9 to 49-fold?in MT45 compared to DSM7^T suggested efficient surfactin assembly and resulted in the overproduction of surfactin.Furthermore,based on the expression trends observed in the transcriptome,there are multiple potential regulatory genes mediating the expression of surfactin synthase.Thus,the results of the present study provide new insights regarding the synthesis and regulation of surfactin in high-producing strain and enrich the genomic and transcriptomic resources available for B.amyloliquefaciens.?5?System metabolic engineering strategy was used to improve the surfactin production of Bacillus subtilis.Firstly,surfactin synthesis of B.subtilis 168 was restored by heterologous expression of sfp gene from B.amyloliquefaciens MT45.Then we constructed a surfactin production platform by deletion of competitive pathway?3.8%of the genome?and overexpression of surfactin transporter and self-resistance associated gene.Surfactin yield was improved from 0.38 g?L^-1 to 3.78 g?L^-1.Subsequently,we overexpressed the genes involved in the branched fatty acids synthesis pathway and heterologous expression of the thioesterase gene tesA from E.coli to provide sufficient precursor for surfactin assembly.Surfactin yield was improved step-by-step from 3.78 g?L^-1 to 9.5 g?L^-1.Finally,the expression level of srf A was significantly improved by integrating comQXPA of MT45 into the equivalent locus of the genome of strain 168 and deleting the potential negative regulator.Surfactin production was further improved to 12.8 g?L^-1,which was the currently reported highest surfactin yield.