State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Department of Applied Biology, School of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Appl Microbiol Biotechnol. 2023 Dec;107(24):7501-7514. doi: 10.1007/s00253-023-12756-1. Epub 2023 Sep 28.
The Actinomycetes Streptomyces lincolnensis is the producer of lincosamide-type antibiotic lincomycin, a widely utilized drug against Gram-positive bacteria and protozoans. In this work, through gene knockout, complementation, and overexpression experiments, we identified LcbR1 (SLINC_1595), a GntR family transcriptional regulator, as a repressor for lincomycin biosynthesis. Deletion of lcbR1 boosted lincomycin production by 3.8-fold, without obvious change in morphological development or cellular growth. The homologues of LcbR1 are widely distributed in Streptomyces. Heterologous expression of SCO1410 from Streptomyces coelicolor resulted in the reduction of lincomycin yield, implying that the function of LcbR1 is conserved across different species. Alignment among sequences upstream of lcbR1 and their homologues revealed a conserved 16-bp palindrome (-TTGAACGATCCTTCAA-), which was further proven to be the recognition motif of LcbR1 by electrophoretic mobility shift assays (EMSAs). Via this motif, LcbR1 suppressed the transcription of lcbR1 and SLINC_1596 sharing the same bi-directional promoter. SLINC_1596, one important target of LcbR1, exerted a positive effect on lincomycin production. As detected by quantitative real-time PCR (qRT-PCR) analyses, the expressions of all selected structural (lmbA, lmbC, lmbJ, lmbV, and lmbW), resistance (lmrA and lmrB) and regulatory genes (lmrC and lmbU) from lincomycin biosynthesis cluster were upregulated in deletion strain ΔlcbR1 at 48 h of fermentation, while the mRNA amounts of bldD, glnR, ramR, SLCG_Lrp, and SLCG_2919, previously characterized as the regulators on lincomycin production, were decreased in strain ΔlcbR1, although the regulatory effects of LcbR1 on the above differential expression genes seemed to be indirect. Besides, indicated by EMSAs, the expression of lcbR1 might be regulated by GlnR, SLCG_Lrp, and SLCG_2919, which shows the complexity of the regulatory network on lincomycin biosynthesis. KEY POINTS: • LcbR1 is a novel and conservative GntR family regulator regulating lincomycin production. • LcbR1 modulates the expressions of lcbR1 and SLINC_1596 through a palindromic motif. • GlnR, SLCG_Lrp, and SLCG_2919 can control the expression of lcbR1.
林肯链霉菌放线菌是林可酰胺类抗生素林可霉素的产生菌，林可霉素是一种广泛用于治疗革兰氏阳性菌和原生动物的药物。在这项工作中，通过基因敲除、互补和过表达实验，我们确定了 GntR 家族转录调节因子 LcbR1（SLINC_1595）是 lincomycin 生物合成的抑制剂。 lcbR1 的缺失使林可霉素的产量提高了 3.8 倍，而形态发育或细胞生长没有明显变化。 LcbR1 的同源物广泛分布于链霉菌属。来自变铅青链霉菌的 SCO1410 的异源表达导致林可霉素产量降低，这意味着 LcbR1 的功能在不同物种中是保守的。 lcbR1 上游序列及其同源物之间的序列比对揭示了一个保守的 16 个碱基对的回文序列（-TTGAACGATCCTTCAA-），通过电泳迁移率变动分析（EMSA）进一步证明这是 LcbR1 的识别模体。通过这个模体，LcbR1 抑制了具有双向启动子的 lcbR1 和 SLINC_1596 的转录。 SLINC_1596 是 LcbR1 的一个重要靶点，对林可霉素的产生有积极的影响。通过定量实时 PCR（qRT-PCR）分析检测到，在发酵 48 小时时，缺失菌株 ΔlcbR1 中所有选定的结构（lmbA、lmbC、lmbJ、lmbV 和 lmbW）、抗性（lmrA 和 lmrB）和调节基因（lmrC 和 lmbU）的表达均上调了 lincomycin 生物合成基因簇，而先前被表征为林可霉素产生调节剂的 bldD、glnR、ramR、SLCG_Lrp 和 SLCG_2919 的 mRNA 量在菌株 ΔlcbR1 中减少，尽管 LcbR1 对上述差异表达基因的调控作用似乎是间接的。此外，通过 EMSA 表明，lcbR1 的表达可能受到 GlnR、SLCG_Lrp 和 SLCG_2919 的调控，这表明 lincomycin 生物合成调控网络的复杂性。要点： • LcbR1 是一种新型保守的 GntR 家族调节剂，调节林可霉素的产生。 • LcbR1 通过回文模体调节 lcbR1 和 SLINC_1596 的表达。 • GlnR、SLCG_Lrp 和 SLCG_2919 可以控制 lcbR1 的表达。
