College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China.
ACS Synth Biol. 2024 Oct 18;13(10):3461-3470. doi: 10.1021/acssynbio.4c00626. Epub 2024 Oct 8.
Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus , which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple species, and the removal of the pathway in the model organism impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as and , by assembling the repressor/operator pair RhaR/ with the well-defined constitutive promoter. Both and exhibited a high level of induced reporter activity, with no leaky expression. has been proven successful for the programmable production of actinorhodin and violacein in . Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other species.
精细调控基因表达对于合成生物技术应用具有重要意义。这对于属来说尤其如此，因为它是多种天然产物的丰富生产者而广为人知。目前，人们对开发有效的基因诱导系统的需求日益增加。在这项研究中，生物信息学分析揭示了多个物种中可能存在的鼠李糖分解代谢途径，并且在模式生物中去除该途径会损害其在以鼠李糖为唯一碳源的最小培养基上的生长。为了阐明分解代谢途径中鼠李糖代谢途径的 LacI 家族转录调节因子 RhaR 的调控机制，进行了电泳迁移率变动分析（EMSA）以鉴定潜在的靶启动子。多重序列比对检索到该途径的 RhaR 操纵子的保守序列（）。然后，采用基于合成生物学的策略，通过组装具有明确组成型启动子的抑制剂/操纵子对 RhaR/，构建了鼠李糖诱导型调控系统，称为和。和都表现出高水平的诱导报告基因活性，没有漏表达。已被证明在生产链霉菌中放线紫红素和紫色素的可编程生产中是成功的。我们的研究扩展了可诱导调控系统的工具包，这些系统将广泛适用于许多其他属。
