In this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d_{(C6HDAP−C4NNADP)} and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDH^{BC621/D120S/W144S/I169P} with 5.32 ± 0.85 U·mg⁻¹ specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

在这项研究中，开发并重建了体内四酶级联途径，用于生产d -对羟基苯基甘氨酸 (D-HPG)，这是一种有价值的中间体，用于生产 β-内酰胺抗生素和精细化学合成。 -酪氨酸。在此途径中，来自谷氨酸棒杆菌的内消旋二氨基庚二酸脱氢酶（ Cg DAPDH）对中间体4-羟基苯乙醛酸的催化转化被确定为限速步骤，随后应用机制引导的“构象旋转”策略来减少氢化物-转移距离 d _{(C6HDAP−C4NNADP)}并增加Cg DAPDH 活性。将蛋白质工程产生的最佳变体（比活性为 5.32 ± 0.85 U·mg ^-1的Cg DAPDH ^{BC621/D120S/W144S/I169P} ）引入设计的途径中，导致 D-HPG 滴度为 42.69 g/L，从 50- g/L L-酪氨酸在 3 L 发酵罐中 24 小时内转化率为 92.5%，分离产率为 71.5%，> 99% 对映体过量。这种四酶级联为从廉价氨基酸工业生产 D-HPG 提供了一种有效的酶促方法。