Optogenetics’ advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.

中文翻译：

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OptoLacI：光遗传学工程乳糖操纵子阻遏物 LacI 对光敏感而不是 IPTG


光遗传学的进步使得光感应因其可微调、可逆性和低毒性的优点而在控制生物过程中具有吸引力。大肠杆菌中常用的乳糖操纵子诱导系统依赖于乳糖或异丙基β-d-1-硫代半乳糖苷（IPTG）与乳糖阻遏蛋白LacI的结合，在控制乳糖操纵子中发挥着关键作用。在这里，我们利用燕麦向光素 1 的光响应光氧电压 2 (LOV2) 结构域作为光控制工具，并将 LacI 改造为两种光响应变体：OptoLacIL 和 OptoLacID。这些变体分别表现出对光和黑暗的直接响应，从而无需 IPTG。在OptoLacI的基础上，我们构建了两个光控大肠杆菌基因表达系统：OptoE.coliLight系统和OptoE.coliDark系统。这些系统通过光操纵实现了大肠杆菌中的双功能基因表达调控，并且与 IPTG 诱导的系统相比显示出优异的可控性。我们将 OptoE.coliDark 系统应用于蛋白质生产和代谢通量控制。蛋白质生产水平与 IPTG 诱导的水平相当。值得注意的是，黑暗诱导产生的 1,3-丙二醇 (1,3-PDO) 和麦角硫因的滴度分别超过 IPTG 诱导产生的 110% 和 60%。 OptoLacI的发展将有助于光遗传学蛋白质工程领域的进步，在各个领域具有巨大的潜在应用前景。