Abstract：
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.
摘要：
5-甲基四氢叶酸(5-MTHF)是在人体中起作用的叶酸的唯一活性形式,并且被广泛用作营养食品。与化学合成的污染不同，微生物合成能够绿色生产5-MTHF。在这项研究中,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和过表达葡萄糖-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，而磷酸戊糖途径的通量增强。NADH/NAD+和NADPH/NADP+比值升高,表明NAD(P)H供应升高。这导致更多的叶酸减少，5-MTHF成功积累至44.57μg/L。随后，表达了来自博伊迪念珠菌和都柏林念珠菌的甲酸脱氢酶，能够催化甲酸钠氧化反应进行NAD（P）H再生。这进一步增加了NAD（P）H供应，导致5-MTHF产量上升至247.36μg/L。此外，为了保持NADH和NADPH之间的平衡，pntAB和stha,编码转肽酶，过度表达。最后,通过在5-MTHF途径中过度表达叶酸的六个关键酶，并在3L发酵罐中采用补料分批培养，菌株Z13达到3009.03μg/L的峰值5-MTHF滴度，到目前为止，大肠杆菌中的最高水平。这项研究是迈向工业规模的微生物5-MTHF生产的重要一步。
