NRPS Gene Analyses And Cloning Of Beauveria Bassiana Under Different Carbon And Nitrogen Sources

Beauveria bassiana is a common entomopathogenic fungus,could produce a variety of secondary metabolites.With the in-depth research on the secondary metabolites of B.bassiana,it is found that the secondary metabolites of B.bassiana have a variety of biological activities.Such as antibacterial,insecticide,inhibition of HIV-1 in vitro,anti-liver cancer,lung cancer,leukemia et al.With metabolic product in the physiological activity are usually non ribosomal peptide(NRPs),to explore the application value of B.bassiana NRPs,the experiment ferment B.bassiana with different carbon and nitrogen source medium,explored the effects of different carbon and nitrogen sources on the antimicrobial activity of their metabolites,as well as the expression levels of 18 NRPS genes.The following results were obtained:Antibacterial activity of B.bassiana extracts in different carbon sources was not as significant as that in nitrogen sources.In the experiment of antibacterial activity of different nitrogen sources,3 drug-resistant bacteria(Staphylococcus aureus,Escherichia coli and Enterococcus faecalis)were only sensitive to extracts from tryptone culture.Pseudomonas aeruginosa was not sensitive to nitrogen sources except casein peptone.When common pathogenic bacteria were used as test bacteria,the antibacterial effect of tryptone extract was significantly higher than that of other nitrogen sources.Different carbon and nitrogen sources had significant effects on the expression of NRPS gene in B.bassiana.Take the NRPS6,NRPS7 and NRPS17 genes as examples.When lactose was the carbon source,the expression of NRPS6 gene was higher than that of other carbon sources.Maltose significantly inhibited the expression of the NRPS6.NRPS7 gene was highly expressed in lactose,but hardly expressed in mannose and maltose.And it was highly expressed in beef powder.NRPS17 gene was highly expressed in lactose,hardly expressed in maltose.Different nitrogen sources had little difference on the expression of NRPS17.The expression ability of NRPS17 from tryptone was slightly stronger than that from other three nitrogen sources.After cloning the full-length cDNA of NRPS17 gene,the bioinformatics analysis results showed that the full length of the ORF of NRPS17 gene cDNA was 4 194 bp,encoding 1 397 amino acid residues.The relative molecular weight of the protein sequence was 153 566.27.And the structural formula was C6865H10797N1813O2073S52.The isoelectric point and fat coefficient were respectively 5.10 and 90.77.In addition,the protein is an unstable protein with an instability coefficient of 42.78 and a half-life of 30 h.BLAST alignment revealed that it contained A-T-C domains.SignalP shows that it is a secretory protein located in the cytoplasmic matrix.Phylogeny showed that the sequence of NRPS17 protein was in the same class as the NRPS of SidC.Therefore,it might be involved in the synthesis of siderophore.This study provided a reference for the functional excavation of the NRPS gene and the search for new active compounds of B.bassiana.