Identification of the Regulon FkbN for Ascomycin Biosynthesis and its Interspecies Conservation Analysis as LAL Family Regulator

Publication date: Available online 17 August 2019Source: Biochemical Engineering JournalAuthor(s): Yue Zhang, Hong Chen, Pan Wang, Jianping WenAbstractAscomycin is a 23-membered polyketide macrolide with high immunosuppressant activity, produced by Streptomyces hygroscopicus var. ascomyceticus. However, the specific regulatory mechanism of the ascomycin biosynthesis pathway remains poorly understood. Here, we show that FkbN is a pathway-specific positive regulator for the ascomycin biosynthesis. Inactivation of fkbN led to a complete loss of ascomycin production ability. Compared with the parental strain, the ascomycin yield of a constructed fkbN overexpression strain increased by 400% to 1800 mg/L. Transcriptional levels of the genes of encoding precursor, PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetases) were blocked in the fkbN in-frame deletion strain and greatly upregulated in the fkbN overexpression strain. Electrophoretic mobility shift assays (EMSAs) revealed that FkbN-DBD protein specifically bound to the intergenic regions of fkbR1-fkbE and fkbB-fkbO. To explore the high conservation among regulators homologous to FkbN, EMSAs showed that the FkbN-DBD protein directly bound to the putative promoters of different macrolide biosynthetic gene clusters. We did further study in the fkbN deletion strain by heterologous genetic complementation with single copy of the homologous LAL family regulators. Results showed that all the heterologous complementa...
Source: Biochemical Engineering Journal - Category: Biochemistry Source Type: research