[Multi-strategy modification for constructing an engineered strain with efficient production of O-acetyl- l-homoserine].

内容摘要

O-acetyl- l- homoserine (OAH) is a key intermediate in the synthesis of various high-value compounds such as l-methionine and has a high market value. However, the production of OAH still faces problems such as low yields and long fermentation periods. In this study, the wild type of the probiotic Escherichia coli Nissle 1917 (EcN) was used as the starting strain. Firstly, we obtained the mutant MetXK185R-G210S-N204T by modifying the key enzyme l-homoserine acetyltransferase (MetX). Further, ppc, asd, and metL were overexpressed to strengthen the synthetic pathways of the precursors l-aspartic acid and l-homoserine. Strategies such as dynamic regulation were adopted to weaken the synthesis of the by-product amino acids. Different intensities of 5{L-End} ' UTR were screened to downregulate the expression level of gltA to balance the supply of another precursor acetyl-CoA, and finally an efficient OAH-producing strain was constructed. The fermentation yield of OAH in the shake flask was 11.18 g/L, and that in the 5 L fermenter reached 63.54 g/L at the time point of 52 h. The strain engineering improved the yield and productivity of OAH and laid a research foundation for the subsequent biological production of l-methionine. O-乙酰- l-高丝氨酸(O-acetyl- l-homoserine, OAH)是 l-蛋氨酸等多种高附加值化合物合成的关键中间体，具有较高的市场价值，但目前OAH生产仍面临产量低、发酵周期长等问题。本研究以大肠杆菌益生菌Escherichia coli Nissle 1917 (EcN)野生型为出发菌株，首先通过对关键合成酶—— l -高丝氨酸乙酰转移酶(l-homoserine O-acetyltransferase, MetX)的改造，获得突变体MetXK185R-G210S-N204T;进一步过表达ppc、asd、metL等基因，强化了前体 l-天冬氨酸和 l-高丝氨酸的合成途径;采用动态调控等策略减弱了副产物氨基酸的合成;并通过筛选不同强度5{L-End} ' UTR，下调gltA基因表达水平，平衡了另一前体乙酰辅酶A的供应，构建了OAH高效生产菌株。最终菌株摇瓶中OAH发酵产量提升至11.18 g/L，5 L发酵罐产量在52 h达到63.54 g/L，本研究提高了OAH的产量和生产效率，为后续 l-蛋氨酸的生物法生产奠定了良好的研究基础。.