The Pleiotropic Regulatory Mechanism Of AdpA_ch In Streptomyces Chattanoogensis

The important industrial natamycin-producer Streptomyces chattanoogensis Lio, which was screened with our independent intellectual property rights, has a very complex secondary metabolic and morphological differentiation process. Although preliminary researches on both have been carried out, the detailed regulatory mechanism is not clear. This study focuses on the regulatory cascade in the natamycin biosynthesis and the cross-regulation network of secondary metabolic and morphological differentiation in S.chattanoogensis L10, in order to provid theoretical basis for enhanced production of natamycin.In our research, we have captured the pleiotropic regulator-AdpAch, which specifically bind to the intergenic region of scnRⅠ-scnRⅡ by DNA affinity. After carried out DNase I Footprinting experiment, we found there are six AdpAch-binding sites located on the intergenic region of scnRⅠ-scnRⅡ, and the function of each AdpAch-binding site is not exactly the same. The transcription of scnRI was activated when AdpAch binds to the Site Ⅲ and Site Ⅳ, and inhibited when AdpAch binds to the Site Ⅰ and Site Ⅱ. Site IV is an activated AdpAch-binding site for the transcription of scnRⅡ, meanwhile, Site V and Site VI are inhibited AdpAch-binding sites for scnRⅡ. These findings lead to the possibility that the pleiotropic regulator AdpAch not only can regulate the transcription of the two pathway-specific regulators at the same time, but also show both positive and negative regulatory ability. The discovery of the positive-negative regulatory ability of AdpAch reveals the artful adaptive mechanism of microbial cells.This study has also examined the self-regulation mechanism of AdpAch. AdpAch has two binding sites on its own promoter. The Competitive experiment showed that AdpAch firstly binds to the Site All to active the transcription of its own promoter. Then, it binds to the Site AI to block its own transcription. Because of the self-regulation, the AdpAch can been stable to a certain amount and the bacteria can grow normally. By reforming the promoter to remove feedback inhibition, we got a high yield strain. Natamycin production was increased by disrupting the Site AI in the promoter of adpAch, almost 1.3 folds(30%) than that of wild-type strain. Base on removing the feedback inhibition, we constructed another higher yield strain YVI by removing the inhibition of AdpAch to the transcription of the pathway-specific regulator, increasing natamycin yield～1.45 fold in comparison with that of wild-type strains. After fermentation on the 15 L bioreactor with glucose feeding, the natamycin yield of YVI was up to 5.4g/L.We have carried out agilent expression microarray to determine the transcription profiles of ΔadpAch and wild-type. In the microarray data, we found that the transcriptional level of wblAch, a whiB-like gene, had severely decreased in the ΔadpAch mutant, and the results of qRT-PCR were shown to coincide with the microarray data. These resulte showed that AdpAch acts as an activator of wblAch transcription in S. chattanoogensis. When carried out EMSA and DNase I Footprinting, we found the AdpAch has a ability to directly bind to the wblAch promoter. The wblAch deletion mutant loses the ability to produce natamycin, and the morphological differentiation was also defective. Because of the low transcriptional level of the whi genes in the ΔwblAch mutant, it is possible that WblAch may play a part in morphological differentiation through interacting with other whi genes. These results indicate the cross-regulation between WblAch and AdpAch in the natamycin biosynthesis and morphological differentiation in S.chattanoogensis L10.