Functional Study Of Uhk01sav Gene In Streptomyces Avermitilis

Streptomyces is a genus of Gram-positive bacteria and possess strong secondary metabolism.It is a prominent producer of a wide variety of antibiotics and other bioactive compounds, which are widely applied in human and veterinary medicine as well as agriculture. So far, researches on regulation of antibiotic biosynthesis in Streptomyces were mostly carried out in the model strain--Streptomyces coelicolor. In our previous study, we showed that deletion of uhk01 gene in S.coelicolor M145, which encodes an unpaired histidine kinase (HK), led to not only obviously increased biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red), but also defective development, including colony color from grey to red, formation of sparse aerial hypha and spores. Bioinformatics analysis shows that uhk01 gene homologues are widely distributed in other different Streptomyces species. In the present study, functional study was carried out to identify the uhk01 homologous gene uhk01sav from an important industrial antibiotic producer-- Streptomyces avermitilis. We found that, similar as the function of uhk01 in S.coelicolor, uhk01sav also play important roles in the regulation of antibiotic biosynthesis and development in S. avermitilis.In S. avermitilis NRRL8165 strain, uhk01sav-deletion mutant (8165â–³uhk01sav) constructed by PCR-targeting system showed increased biosynthesis of oligomycin A, approximately 5 to 6-fold higher than that of the wild type. Moreover, the mutant grew slower and showed defective sporulation when compared with NRRL8165. The increased synthesis of oligomycin A and defective sporulation could be readily rescued by complementation with uhk01sav gene, suggesting that uhk01sav was a negative regulator for oligomycin biosynthesis and had a close relationship with sporulation. In order to learn more about the molecular mechanism by which uhk01sav exerted on oligomycin biosynthesis and the effects on other two important antibiotic biosynthetic pathway, real-time RT-PCR was performed to analyze the transcriptional profiles of the wild type strain NRRL8165 and its mutant 8165â–³uhk01sav. The results showed that regulation of uhk01sav on oligomycin biosynthesis was mediated by pathway-specific regulators, OlmRI and OlmRII. Meanwhile, deletion of uhk01sav also affected the biosynthesis of both avermecin and pentalenolactone.In cross-complementation assay, we found, phenotype changes of the mutants S.avermitilis 8165â–³uhk01sav and S.coelicolor M145â–³uhk01 could be complemented with the respective wild-type genes and also their orthologue genes. Antibiotic production and sporulation of the complemented strains resembled as the wild-type strains, suggesting that uhk01sav and uhk01av shared the similar functionality.As mentioned above, uhk01 and its analogs might be conserved regulators involved in the regulation of antibiotics biosynthesis and morphological differentiation in Streptomyces and they should be the useful target genes for the metabolic engineering of important industrial Streptomyces to improve antibiotic biosynthesis.