Characterization Of Surfactin Synthesis Genes And Dissolved Oxygen Regulatory Pathways In Bacillus Velezensis CMT-6

Bacillus velezensis species is closely related to Bacillus amyloliquefaciens and Bacillus subtilis.These three bacilli species are involved in the surfactin bio-synthesis.Surfactin is a key constituent of antimicrobial lipopeptide with strong surfactivity,excellent antibacterial activity and good foamability and emulsifiability.Therefore,surfactin plays an important role in many fields including food,medicine and cosmetic.Normally,surfactin is produced by bacteria only at low concentrations,and the reason is still not known,it impedes the large-scale application process of surfactin seriously.In addition,increasing the rotational speed is used to improve dissolved oxygen concentration in industrial microbiological fermentation processes to induce excessive surfactin nowadays.However,surfactin wastage increased due to the large amount of foam created by rapid stirring.So the production of surfactin relies deeply on oxygen availability.So,it is a challenge to identify the surfactin-linked genes in B.velezensis CMT-6 and analyze gene expression in different dissolved oxygen environments to determine crucial regulatory pathways.In this dissertation,the whole genome sequencing was used to analysis the genomic information and to excavate the surfactin-linked genes of Bacillus velezensis CMT-6 possessing high surfactin production.Meanwhile,comparative genomes of four bacilli strains with different surfactin production capacities were conducted to reveal the reason of the different surfactin synthesis abilities,and to analysis the characteristics of surfactin synthesis genes in CMT-6.Transcriptome technology was further used to investigate crucial genes and regulatory pathways responding to dissolved oxygen.These could establish a theoretical basis and new ideas for efficient screening of natural strains with high productivity and improving the surfactin production in low oxygen environment.The main research protocols and results are described below.1.The whole genome of CMT-6 was sequenced using the second generation and third generation sequencing technology.The lipopeptides formed during fermentation were detected by TOF-MS.The genome comprised of a single circular chromosome(3,928,489bp),with an overall G+C content of 46.55%.All the genes annotated in Cluster of Orthologus Gene(COG)database were mainly involved in amino acid and carbohydrate transport and metabolism,and secondary metabolite biosynthesis,transport and catabolism.The genes annotated in Kyoto Encyclopaedia of Genes and Genomes(KEGG)database participated in signal transduction and carbonhydrate metabolism.When annotated in Gene Ontology(GO)database,most genes were associated with catalytic activity,and metabolic and cellular processes.These genes facilitated CMT-6 to respond to environmental changes efficiently,supply nutrients for growth,and provide precursors for surfactin synthesis.CMT-6 possessed an integrated surfactin synthetase gene,srfAA-AD.Most CMT-6 genes were associated with surfactin than iturin and fengycin synthesis.More surfactin components(m/z:994.6,1008.6,1009.6,1010.6,1022.6,1036.6)were detected than iturin(m/z:1044.6,1058.6,1059.6)and fengycin(m/z:1434.6)during fermentation.The lipopeptide abundance positively correlated with the surfactin,iturin anf fengycin genes,which is the main reason for CMT-6 to excessive accumulation of surfactin.2.Comparative genomic analysis between B.velezensis CMT-6,B.subtilis 168,B.amyloliquefaciens DSM7~Tand B.velezensis FZB 42 was conducted.CMT-6 and FZB 42exhibited a conservative sequence structure and a close relationship.DSM7~Tranked second and CMT-6 was farthest to the 168 strain.The co-linear relationship among various strains was in agreement with their mutual relationships.And surfactin synthesis could be affected by these two factors.CMT-6 shared 5 surfactin related genes with FZB42 and DSM7~T,and shared 3 related genes with the lowest identify values with the 168 strain.The ability to produce surfactin was influenced by the core genes in different strains and the identify values.Comparing CMT-6 with FZB 42,only non-synonymous mutations were detected in the synthetase gene srfAC.Non-synonymous mutations were identified in synthetase genes srfAA,srfAB and srfAC,and other related genes spoVT and cody in CMT-6 and DSM7~T.Besides these genes with non-synonymous mutations,a premature stop codon was detected in srfAC in the168 strain.The specific selection of amino acids could be changed by synthase encoded by the synthetase gene with non-synonymous.And the strain owned synthase gene with premature stop codon was incapable of synthesizing surfactin.3.The thallus of CMT-6 were collected during low-oxygen(agitation 50 rpm),mid-oxygen(agitation 100 rpm)and high-oxygen supply(agitation 200 rpm)and RNA was extracted to study the transcript profiles in order to identify the crucial genes and pathways related to surfactin in response to oxygen.The transcriptome data analysis revealed that the largest number of differential expressed genes(DEGs)which were mainly annotated in two GO items(bacterial-type flagellum-dependent cell motility and bacterial-type flagellum basal body)and in metabolic pathways were detected in low-and mid-oxygen supply studies.Comparing the transcriptome of mid-oxygen supply with that of high-oxygen supply,DEGs were mainly assigned in 7 GO items(especially in the oxidation-reduction process),and enriched as microbial metabolism in diverse environments,carbon metabolism and two component system(TCS)to KEGG database.The expression of genes involved in the pathways of TCA,fatty acid biosynthesis,TCS and protein export were useful to encode more precursors,resistance to surfactin and excretion of surfactin to synthesize high surfactin production in high oxygen supply.Synthetase gene regulated by rne5 including srfAA,srfAC and srfAD negatively correlated with the surfactin yield.Other five genes srfAB,spoVT,AbrB,comP and rapC positively correlated with the surfactin yield.