Screening An Epiphytic Bacterium Producing Surfactin From Grapes And Heterologous Expression Of Its Biosynthetic Gene Clusters

Lipopeptide metabolites,with advantages of wide antimicrobial spectrum,active stability and difficult to form drug resistance,have a broad application prospect in food,feed,agriculture and medical field.The traditional research methods on microbes producing lipopeptide are restricted to chemical screening,bioactive screening or combined with both.However,these methods can't predict the type of secondary metabolites and always lead to repetition of known compounds,wasting time and labour.In recent years,with the rapid development of the molecular biology,the increased number of bacterial genomic sequence and the mechanism of secondary metabolites have be illuminated.Directional screening method based on the probe of nonribosomal peptide synthetases(NRPSs)conserved genes,is able to predict the type of metabolites by PCR amplification and phylogenetic analysis.The screening method targeting on the gene of peptide compounds,is one of the important strategy to study the natural secondary metabolites.In this study,strain producing surfactin was screened from grapes based on A domain in NRPSs fountional genes by PCR amplification.Moreover,the nucleotide sequence information of surfactin biosynthetic gene cluster from the strain was obtained by the whole genome sequence analysis.On this basis,the surfactin biosynthetic gene cluster was cloned by transformation-associated recombination(TAR),CRISPR/Cas9 and their intercombination.(1)Five epiphytic bacteria named as PTWA1?5,were isolated from "summer black" grapes using plate streaking and slant culture methods.Combined with the results of 16SrDNA gene and the physiological and biochemical characteristics,five epiphytic bacteria were identified as Bacillus flexus?Bacillus subtilis.Bacillus rhizosphaerae?Kocuria marina?Pseudomonas jessenii.And B.subtilis predicted to produce surfactin was screened out based on NRPSs functional genes.(2)The shuttle vector which can specially cloned surfactin biosynthetic gene cluster was constructed in Saccharomyces cerevisiae.The skeleton plasmid of pLLX13,maker bla/CYH2 from pLLX8,and genomic homologous arms from B.subtilis were assembled into yeast caputre vector(YCV)through the efficient mechanism of homologous recombination from S.cerevisiae CRY 1-2.(3)Then we cloned Surfactin biosynthetic gene cluster with combinatorial methods:1)enzyme restriction technique combined with TAR;2)CRISPR/Cas9 editing technique combined with TAR;3)CRISPR/Cas9 combined with Gibson assembly.Finally the recombinant vector we screened by gel electrophoresis test,was heterologously induced expression by ITPG in Escherichia coli BL21.The crude extract was obtained from the fermented broth and mycelia of the E.coli BL21 by solvent extraction and was analyzed with thin layer chromatography?high performance liquid chromatography and electrospray mass spectrometry.Compared with control group,samples from recombinant clones might contain the compound of Surfactin.As a result,a pure compound was obtained from the fermentation product of the positive recombinant clones by solvent extraction and silica gel column chromatography,and its chemical sturture was identified as surfactin A by nuclear magnetic resonance.It seems that we successfully cloned and heterologous expressed Surfactin biosynthesis gene cluster E.coli.Study of vector construction and heterologous expression of surfactin gene cluster with three methods in this work provide theoretical basis and technical support for improving the technology system in synthetic biology.