代谢工程改造大肠杆菌高产O-乙酰高丝氨酸的关键问题研究

O-Acetylhomoserine is a key precursor for L-methionine production by bioconversion, and L-methionine is widely used in the fields of feed industry, food industry and medicine. Up to now, the efficient biosynthesis of O-acetylhomoserine is hardly achievable. In our earlier study, the biosynthetic pathway of O-acetyl-homoserine in Escherichia coli was constructed, and the feedback inhibition of homoserine acetyltransferase by O-acetylhomoserine was first discovered. However, the molecular mechanism of feedback inhibition by O-acetylhomoserine is unclear. Furthermore, the need for large quantities of reducing power and co-factors, competing metabolic pathways and complex metabolic regulation are the limit factors for the efficient production of O-acetylhomoserine. In this study, the feedback-inhibition resistance and stability of homoserine acetyltransferase will be improved by protein engineering, and the mechanism of anti-feedback will be analyzed by homology modeling. Furthermore, the biosynthesis pathway of O-acetylhomoserine will be systematically optimized by increasing the metabolic flow, optimizing the biosynthesis of reducing power and cofactors, and an efficient strain for O-acetylhomoserine production will be constructed. These results will not only elucidate the anti-feedback mechanism of homoserine acetyltransferase, but also provide a good foundation for L-methionine production by bioconversion.

O-乙酰高丝氨酸是生物转化法合成L-甲硫氨酸的关键前体化合物，而甲硫氨酸在饲料、食品、医药等领域具有广泛的用途。目前，O-乙酰高丝氨酸难以高效地生物合成。前期我们在大肠杆菌构建了O-乙酰高丝氨酸合成途径，首次发现O-乙酰高丝氨酸的合成受高丝氨酸乙酰转移酶的反馈抑制，有关该反馈抑制机理尚不明确。此外，大量还原力及辅因子的需求、竞争支路的存在以及复杂的代谢调控是O-乙酰高丝氨酸高效合成的限制因素。本研究拟对高丝氨酸乙酰转移酶进行蛋白改造，提高其抗O-乙酰高丝氨酸反馈抑制的能力及稳定性，并通过同源建模分析突变位点与抗反馈之间的关系；通过系统优化O-乙酰高丝氨酸代谢途径，包括提高O-乙酰高丝氨酸合成代谢流、优化还原力和辅因子的生物合成，获得O-乙酰高丝氨酸高产菌株。相关工作不仅可以阐明高丝氨酸乙酰转移酶抗O-乙酰高丝氨酸反馈抑制机理，同时也为生物转化法生产L-甲硫氨酸奠定良好基础。

OAH是一种具有潜在工业应用价值的前体化合物，是生物转化法合成L-甲硫氨酸的关键原料。但是由于其生物合成途径复杂，关键途径酶活性受到严谨反馈抑制效应，目前微生物难以高效合成O-乙酰高丝氨酸。本研究通过蛋白质工程等策略对高丝氨酸乙酰转移酶进行定向改造，获得了多个酶活性及抗O-乙酰高丝氨酸反馈性能提升的有益突变体，同时结合系统代谢工程方法对代谢途径进行组合优化，实现了大肠杆菌O-乙酰高丝氨酸的高效合成，最终获得的工程菌株能够发酵生产62.7g/L的O-乙酰高丝氨酸。相关研究可以为生物转化法生产下游产品L-甲硫氨酸奠定良好基础。