Preliminary Research On Regulatory Genes In The Enduracidin Gene Cluster

Enduracidin that is catalyzed by Non-ribosomal Peptides Synthetase is a lipopeptide antibiotic produced by Streptomyces fungicidicus.Several researches have revealed that enduracidin can inhibit the activity of Gram-positive bacteria well.Meanwhile,it is also resistant to methicillin-resistant Staphylococcus aureus(MRS A)and vancomycin-resistant Enterococcus faecium(VRE).Moreover,it has no cross resistance with the existing antibiotics.However,due to its poor water solubility,enduracidin is usually used in the livestock farm.The addition of enduracidin to fodder can promote animal growth and enhance economic benefits.In addition,the long fermentation period and low productivity also limit the application of enduracidin.Thus,it is of significance to find proper solutions to the low productivity and high cost problems.Ribosome engineering or metabolic engineering methods are two major ways to obtain the strains with high productivity.According to the literatures,some researchers have already used the ribosome engineering method to improve the enduracidin titer.However,the regulatory mechanism of microorganisms to produce enduracidin has not yet been clarified..This study screened the fermentation medium of MRSA to synthesize enduracidin,followed by exploring the conjugational transfer factors.Besides,the functionalities of regulatory gene orf22 and two-component regulatory gene orf41,orf42,orf43 were studied,so as to improve the titer of enduracidin by metabolic engineering method.This work focused on the following aspects:1.Screening of the fermentation media of Streptomyces fungicidicus to produce enduracidin and establishing the genetic manipulation method.Various medium reported in the literature and collected in our lab were tested for the optimization of culture medium.An optimal fermentation medium(Rsa)was obtained with the optimal temperature and time of 28 oC and 8d.Under this condition,the enduracidin yield was 0.67±0.25 mg/L.Moreover,the genetic manipulation system of Streptomyces fungicidicus was established by trying different heat shock time and different spore concentrations.The optimal spore concentration was 108 cell/mL and the heat shock time was 30 min.2.Functionality of orf22 in the enduracidin biosynthetic gene cluster.A putative SARP family protein encoded gene orf22 was found in the enduracidin gene cluster by bioinformatics analysis.Amino acid sequence alignment results indicated that Orf22 shared 35%amino acids identity to SARP family StrR.However,Orf22 lacked N terminal HTH domain,as well as the activation domain which were indispensable for the SARP family.StrR was reported to be a positive regulator in the biosynthesis of streptomycin.Thus,it was assumed that Orf22 was also a positive regulator.To study its functionality,the overexpression and inactivation of orf22 were carried out.The enduracidin yield increased by 250%when orf22 was overexpressed compared to that of wild type strain.The Aorf22 mutant lost the ability to produce enduracidin.3.Functionality of orf41,orf42,and orf43 in the enduracidin biosynthetic gene cluster.Besides a putative SARP gene,there were another three genes,which may belong to two-component regulatory genes in the enduracidin biosynthetic gene cluster.The enduracidin yield declined by 97%when orf41 was overexpressed.Gene orf41 was considered as a negative regulatory gene.The enduracidin yield increased by 97%once orf42 was overexpressed,implying that orf42 up-regulated the biosynthesis of enduracidin.The enduracidin yield did not change significantly when orf43 was overexpressed,indicating that orf43 may have no influence on the production of enduracidin.