Function Of The Regulator AniF In Anisomycin Biosynthesis

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi.It has been successfully used in the treatment of amoeba protozoa and trichomonas vaginalis and it has potential antitumor activity.Moreover,it is one of the important effective components in Agricultural Antibiotic120,which has been widely used as naturally-originated agents for treatment of crop decay in China,such as crop powdery mildew,watermelon rot and rice sheath blight.Therefore,anisomycin has attracted the interest of many scientists since its discovery in 1954.The chemical synthesis of anisomycin has recently been reported,but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts.The biosynthetic gene cluster(BGC)of anisomycin in Streptomyces hygrospinosus var.beijingensis has been identified in our previous work,while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S.hygrospinosus var.beijingensis.In this study here,it was proved by gene complementary and overexpression that AniF,a transcriptional regulator of LuxR family,was essential for activating anisomycin biosynthesis,and that overexpression strain could increase the yield of anisomycin and its derivatives.The yeild of anisomycin,deactylanisomycin,component III and component IV in overexpression strain was improved up to 1.2,2.9,2.1 and 3.4 times higher than that in WT strain,respectively.According to the bioinformatics analysis,we speculated that AniF belongs to the atypical response regulators(ARRs).Then RT-PCR proved that anisomycin biosynthetic gene cluster(BGC)contains two transcriptional units,and real-time quantitative PCR(RT-qPCR)revealed AniF acted on the promoter of operon aniR-G,and positively regulated the transcription of the anisomycin BGC.Finally,one AniF-binding site in the promoter region of aniR was identified by EMSA and DNase I footprinting assay and an inverted repeat sequence(5’-GGGC-3’)composed of two 4-nt half sites in the protected region was found.In addition,the small molecular signal competition experiments showed that anisomycin and the four intermediates of biosynthesis pathway had no effect on the binding function of AniF.In conclusion,based on the previous studies of anisomycin biosynthesis,the regulatory mechanism of AniF was revealed by gene complemention and overexpression,bioinformatics analysis,RT-PCR,RT-qPCR,EMSA and DNase I footprinting.These results provide new insights of anisomycin biosynthesis and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives.