The ROK‐family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis

Abstract

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5 0-TTKAMKHSTTSAV-3 0 , was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.

Effects of dual deletion ofglnRandmtrAon expression of nitrogen metabolism genes inStreptomyces venezuelae

“…The concentration of the crude lysates was determined by the Pierce Protein Assay Kit. Equal amounts of crude lysates were separated by SDS‐PAGE (12%) and then transferred to Hybond‐ECL membranes (GE Amersham), which were blocked with 5% fat‐free milk at room temperature for 2 hours, washed twice and incubated with anti‐FLAG mAb (1:3000; Boster Biological Technology) at 4°C overnight (Lu et al ., 2020b; Yan et al ., 2020). Next, the membranes were washed twice before incubating with the HRP‐conjugated goat anti‐mouse IgG (H + L) secondary antibody (1:5000; Boster Biological Technology) for 50 min at room temperature.…”

Carbon catabolite regulation of secondary metabolite formation, an old but not well‐established regulatory system

“…In this and other analyses, Rok7B7 (a member of the ROK family regulators) and XylFGH (the xylose uptake operon) were upregulated by glucose through an independent way of Glk (Romero-Rodr ıguez et al, 2016a). In S. coelicolor Rok7B7 negatively regulates the adjacent operon xylFGH (Swiatek et al, 2013;Lu et al, 2020), promotes ACT production and suppresses both RED and CDA biosynthesis (Fig. 2), probably by direct binding to actII-ORF4 and redZ promoters (Park et al, 2009).…”

“…This signal molecule acts as a negative allosteric effector of DasR (Fig. 1), a GntR family transcriptional factor that controls the GlcNAc transport genes, morphological differentiation and secondary metabolism (Rigali et al, 2006;Magdalena et al, 2012). Its targets include the antibiotic regulatory gene actII-ORF4 (van Wezel and McDowall, 2011) and some genes related to the synthesis of coelimycin and CDA (Swiatek-Polatynska et al, 2015).…”

“…This carbon source, in turn, acts as an inducer releasing the promoter from binding by Rok7B7. Additionally, eleven new target genes of Rok7B7 involved in the development, secondary metabolism, glucose uptake and primary metabolic processes have been identified in this microorganism (Lu et al, 2020) (Fig. 2).…”

“…In S . coelicolor Rok7B7 negatively regulates the adjacent operon xylFGH (Swiatek et al ., 2013 ; Lu et al ., 2020 ), promotes ACT production and suppresses both RED and CDA biosynthesis (Fig. 2 ), probably by direct binding to actII‐ORF4 and redZ promoters (Park et al ., 2009 ).…”