AflQ1-Q2 represses lincomycin biosynthesis via multiple cascades in Streptomyces lincolnensis.

Lincomycin is a broad-spectrum antibiotic and particularly effective against Gram-positive pathogens. Albeit familiar with the biosynthetic mechanism of lincomycin, we know less about its regulation, limiting the rational design for strain improvement. We therefore analyzed two-component systems (TCSs) in Streptomyces lincolnensis, and selected eight TCS gene(s) to construct their deletion mutants utilizing CRISPR/Cas9 system. Among them, lincomycin yield increased in two strains (Δ3900-3901 and Δ5290-5291) while decreased in other four strains (Δ3415-3416, Δ4153-4154, Δ4985, and Δ7949). Considering the conspicuous effect, SLINC_5291-5290 (AflQ1-Q2) was subsequently studied in detail. Its repression on lincomycin biosynthesis was further proved by gene complementation and overexpression. By binding to a 16-bp palindromic motif, the response regulator AflQ1 inhibits the transcription of its encoding gene and the expression of eight operons inside the lincomycin synthetic cluster (headed by lmbA, lmbJ, lmbK, lmbV, lmbW, lmbU, lmrA, and lmrC), as demonstrated by quantitative RT-PCR and electrophoretic mobility shift assays. Besides, the regulatory genes including bldD, glnR, lcbR1, and ramR are also regulated by the TCS. According to the screening towards nitrogen sources, aspartate affects the regulatory behavior of histidine kinase AflQ2. And in return, AflQ1 accelerates aspartate metabolism via ask-asd, asd2, and thrA. In summary, we acquired six novel regulators related to lincomycin biosynthesis, and elucidated the regulatory mechanism of AflQ1-Q2. This highly conserved TCS is a promising target for the construction of antibiotic high-yield strains. KEY POINTS: • AflQ1-Q2 is a repressor for lincomycin production. • AflQ1 modulates the expression of lincomycin biosynthetic and regulatory genes. • Aspartate affects the behavior of AflQ2, and its metabolism is promoted by AflQ1.