New targets of TetR-type regulator SLCG_2919 for controlling lincomycin biosynthesis in Streptomyces lincolnensis.

ABSTRACT

The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.