Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is difficult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA, menG, and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P43 resulted in 8.1 ± 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 ± 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr, and ispD-ispF genes in MEP module with strong promoter P43 increased the titer of MK-4 to 78.1 ± 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaK1, mvaK2, mvaD, mvaS, and mvaA) resulted in 90.1 ± 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA, and menG in menaquinone pathway with strong promoter P43 increased the titer of MK-4 to 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future.

中文翻译：

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用于改进枯草芽孢杆菌中甲基萘醌 4 生物合成的组合工程

Menaquinone-4 (MK-4) 是维生素 K 的一种形式，在心血管和骨骼健康方面发挥着重要作用。Menaquinone-4 (MK-4) 是一种珍贵的维生素 K2，很难有机合成，现在主要通过微生物发酵生产。在此，我们通过枯草芽孢杆菌 168 中的组合途径工程显着提高了 MK-4 的合成效率，枯草芽孢杆菌 168 是一种广泛用于生产营养品的模型工业菌株。与MK-4合成相关的代谢网络包括四个模块，即MK-4生物合成模块、甲基赤藓糖醇磷酸（MEP）模块、甲羟戊酸依赖性（MVA）类异戊二烯模块和甲基萘醌模块。来自集胞藻属的 menA、menG 和 crtE 基因的过表达。具有强组成型启动子 P43 的 MK-4 合成模块中的 PCC 6803 产生 8.1 ± 0。2 mg/L MK-4（在野生型枯草芽孢杆菌 168 中未检测到 MK-4）。通过敲除 hepT 基因，MK-4 滴度进一步增加了 3.8 倍，达到 31.53 ± 0.95 mg/L，该基因催化二磷酸法呢酯转化为二磷酸庚烯酯。此外，在具有强启动子 P43 的 MEP 模块中同时过表达 dxs、dxr 和 ispD-ispF 基因使 MK-4 的滴度增加到 78.1 ± 1.6 mg/L。此外，异质 MVA 模块基因（mvaK1、mvaK2、mvaD、mvaS 和 mvaA）的表达导致 90.1 ± 1.7 mg/L 的 MK-4。最后，为了进一步将增强的碳代谢通量转化为 MK-4，在具有强启动子 P43 的甲基萘醌途径中同时过表达基因 crtE、menA 和 menG 使 MK-4 的滴度增加至 120.1 ± 0.6 mg/L摇瓶和 145 ± 2.8 mg/L 在 3-L 分批补料生物反应器中。