5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD⁺ and NADPH/NADP⁺ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 μg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 μg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 μg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

中文翻译：

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工程氧化还原辅因子平衡可提高大肠杆菌中 5-甲基四氢叶酸的产量

5-甲基四氢叶酸 (5-MTHF) 是叶酸在人体内发挥作用的唯一活性形式，被广泛用作营养保健品。与化学合成带来的污染不同，微生物合成可以实现5-MTHF的绿色生产。本研究选择大肠杆菌BL21（DE3）作为宿主。最初，通过删除6-磷酸果糖激酶1并过表达葡萄糖-6-磷酸1-脱氢酶和6-磷酸葡萄糖酸脱氢酶，糖酵解途径通量降低，而磷酸戊糖途径通量增强。 NADH/NAD ⁺和 NADPH/NADP ⁺的比率增加，表明 NAD(P)H 供应增加。这导致更多的叶酸减少，并且 5-MTHF 成功积累至 44.57 μg/L。随后，来自博伊丁假丝酵母和都柏林假丝酵母的甲酸脱氢酶被表达，它们能够催化甲酸钠氧化反应以再生NAD(P)H。这进一步增加了 NAD(P)H 的供应，导致 5-MTHF 产量上升至 247.36 μg/L。此外，为了维持NADH和NADPH之间的平衡，编码转氢酶的pntAB和sthA被过表达。最后，通过过表达叶酸至 5-MTHF 途径中的六种关键酶并在 3 L 发酵罐中采用补料分批培养，菌株 Z13 的 5-MTHF 滴度峰值达到 3009.03 μg/L，这是大肠杆菌中报道的最高水平。迄今为止。这项研究是迈向工业规模微生物 5-MTHF 生产的重要一步。