State Key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.
J Basic Microbiol. 2023 Feb;63(2):168-178. doi: 10.1002/jobm.202200488. Epub 2022 Oct 25.
l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.
l-高丝氨酸是一种非蛋白质氨基酸，用于合成工业中的许多活性物质。在这里，为了开发出一种强大的 l-高丝氨酸生产菌株，我们使用大肠杆菌 W3110 作为底盘进行工程改造。基于之前构建的阻断 l-高丝氨酸合成竞争途径的构建体，我们通过启动子替换策略过表达了五个基因，以提高 l-高丝氨酸的产量，包括增强 l-高丝氨酸合成的前体（ppc、thrA 和 asd）、加强 NADPH 供应（pntAB）和外排转运蛋白（rhtA），以提高 l-高丝氨酸的产量。然而，质粒丢失是造成工程菌株发酵性能剧烈波动的原因，产量在 2.1 到 6.2 g/L 之间。然后，将hok/sok 毒素/抗毒素系统引入游离质粒表达盒中，以维持附加体质粒的遗传稳定性；因此，质粒丢失率急剧下降，导致工程菌株 SHL17 在生产 l-高丝氨酸方面表现出优异的稳定性，在摇瓶中达到 6.3 g/L，在 5-L 发酵罐中无需添加抗生素即可达到 44.4 g/L。这项工作验证了 hok/sok 毒素/抗毒素系统与启动子工程相结合，在不使用抗生素的情况下，有效地提高了大肠杆菌附加体质粒的遗传稳定性。
