Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Bioresour Technol. 2023 Dec;390:129862. doi: 10.1016/j.biortech.2023.129862. Epub 2023 Oct 13.
Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysC from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.
章鱼胺是一种天然保护剂，由一些在低浓度下自然合成的极端环境微生物产生，这些微生物通常难以培养。因此，需要开发一种高效、环保的章鱼胺生产工艺。本研究利用来源于盐单胞菌（Halomonas venusta）的 ectABC 基因簇，在大肠杆菌 BL21（DE3）中实现了 1.7 g/L 的章鱼胺产量。通过优化表达质粒，产量提高到了 2.1 g/L。此外，还过表达了来自谷氨酸棒杆菌（Corynebacterium glutamicum）的天冬氨酸激酶突变体 LysC 和来自盐沼盐单胞菌（Halomonas elongata）的天冬氨酸半醛脱氢酶，以增加前体供应。进一步对半醛还原酶 EctB 进行半理性设计，E407D 突变提高了 13.8%的章鱼胺产量。为了提高乙酰辅酶 A 的供应，引入了非氧化磷酸戊糖途径。最终，优化后的菌株 ECT9-5 通过分批补料发酵生产了 67.1 g/L 的章鱼胺，葡萄糖得率为 0.3 g/g，所得动力学模型拟合度良好。
