Huang Wei, Li Jinlong, Zhu Qiyao, Lv Jianan, Zhu Rui, Pu Chunxiang, Zhao Huabing, Fu Gang, Zhang Dawei
School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
J Agric Food Chem. 2024 Oct 7;72(41):22672-81. doi: 10.1021/acs.jafc.4c07385.
As an indispensable member of the family of lipid vitamins, vitamin K (MK-7) plays an important role in blood coagulation, cardiovascular health, and kidney health. Microbial fermentation is favored due to its high utilization rate of raw materials, simple operation, and moderate conditions. However, the biosynthesis pathway of vitamin K in microorganisms is highly complex, which hinders its industrial production in microbial cell factories. One of the major challenges is the stable expression and deregulation of key enzymes in the vitamin K biosynthesis pathway, which remains unclear and has undergone little investigation. In this study, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase (MenA) were identified as pivotal enzymes in the biosynthesis of vitamin K. To investigate the catalytic efficiency of MenD in the biosynthesis pathway of vitamin K, structure-based mutation design and site-directed mutagenesis were performed. Three mutation sites were identified in MenD: A115Y, R96 M, and R323M, which improve the expression level and protein stability. Meanwhile, the MenA mutant T290M, which exhibits improved protein stability, was obtained by modifying its hydrophobic stacking structure. Finally, an engineered strain noted ZQ13 that combinatorially overexpressed MenD (A115Y) and MenA (T290M) mutants was constructed and achieved 338.37 mg/L vitamin K production in a 3-L fermenter.
作为脂溶性维生素家族中不可或缺的一员，维生素K（MK-7）在血液凝固、心血管健康和肾脏健康方面发挥着重要作用。微生物发酵因其原料利用率高、操作简单、条件温和而备受青睐。然而，微生物中维生素K的生物合成途径高度复杂，这阻碍了其在微生物细胞工厂中的工业化生产。主要挑战之一是维生素K生物合成途径中关键酶的稳定表达和调控，目前仍不清楚且研究较少。在本研究中，2-琥珀酰-5-烯醇丙酮酸-6-羟基-3-环己烯-1-羧酸合酶（MenD）和1,4-二羟基-2-萘甲酸聚异戊二烯基转移酶（MenA）被确定为维生素K生物合成中的关键酶。为了研究MenD在维生素K生物合成途径中的催化效率，进行了基于结构的突变设计和定点诱变。在MenD中鉴定出三个突变位点：A115Y、R96M和R323M，这些位点提高了表达水平和蛋白质稳定性。同时，通过修饰其疏水堆积结构获得了蛋白质稳定性提高的MenA突变体T290M。最后，构建了组合过表达MenD（A115Y）和MenA（T290M）突变体的工程菌株ZQ13，并在3-L发酵罐中实现了338.37 mg/L的维生素K产量。
