光遗传学的进步使光诱导由于其精细可调性的优势而对控制生物过程具有吸引力,可逆性,和低毒性。乳糖操纵子诱导系统,常用于大肠杆菌,依赖于乳糖或异丙基β-d-1-硫代吡喃半乳糖苷(IPTG)与乳糖抑制蛋白LacI的结合,在控制乳糖操纵子中起着关键作用。这里,我们利用来自Avenasativa光效素1的光响应光氧电压2(LOV2)域作为光控制工具,并将LacI设计为两个光响应变体,OptoLacIL和OptoLacID。这些变体表现出对光明和黑暗的直接反应,分别,消除对IPTG的需要。建立在OptoLacI之上,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。这些系统通过光操作实现大肠杆菌中的双功能基因表达调节,并且与IPTG诱导的系统相比显示出优异的可控性。我们使用了OptoE。大肠杆菌系统对蛋白质生产和代谢通量的控制。蛋白质生产水平与IPTG诱导的水平相当。值得注意的是,黑暗诱导的1,3-丙二醇(1,3-PDO)和麦角硫因产生的滴度超过IPTG诱导的110%和60%,分别。OptoLacI的发展将有助于光遗传学蛋白质工程领域的发展,在各个领域拥有大量潜在的应用。
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.