Identification Of The Regulon FkbN For Ascomycin Biosynthesis And Its Conservative Analysis As LAL Family Pathway-specific Regulator

Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant activity,produced by Streptomyces hygroscopicus var.ascomyceticus.However,its biosynthesis regulon remains largely unknown.This paper focuses on the specific regulator FkbN in the ascomycin synthesis gene cluster and on the interspecies conservation of LAL family regulators.Bioinformatics analysis revealed that FkbN belongs to the LAL family transcriptional regulator.Inactivation of fkb N led to a complete loss of ascomycin production ability.Compared with the parent strain,the ascomycin yield of a constructed fkb N overexpression strain increased by 400% to 1800 mg/L.Therefore,the regulatory factor encoded by the fkb N gene is capable of positively regulating ascomycin biosynthesis.Eight transcription units were identified in the ascomycin biosynthesis cluster.Transcriptional analysis showed that most of the genes were blocked in the fkb N in-frame deletion strain and greatly upregulated in the fkb N overexpression strain.Electrophoretic mobility shift assays(EMSAs)revealed that FkbN with an intact C-terminal DNA binding domain specifically bound to the intergenic region of fkb R1-fkb E and fkb B-fkb O.Furthermore,to explore whether there is a high conservation among regulators homologous to FkbN,the protein was shown to directly bind to putaive promoters from similar macrolide biosynthetic clusters by EMSAs.Accordingly,heterologous complementation strains with single copies of the homologous LAL family regulators,such as ave R(from S.avermitilis),rap H(from S.hygroscopicus)or fkb N(from S.tsukubaensis),restored ascomycin production.Introduction of a single copy of fkb N into the tacrolimus producing strain S.tsukubaensis or rapamycin producing strain S.hygroscopicus,both enhanced the yields of the corresponding antibiotics,thus demonstrating the functional conservation among these LAL regulators,which appear to be fully exchangeable.