Lu Chuanchuan, Liu Yanfeng, Li Jianghua, Liu Long, Du Guocheng
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, P.R. China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, P.R. China.
J Microbiol Biotechnol. 2019 Jan 28;31(1):154-162. doi: 10.4014/jmb.1910.10069.
L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 μg/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 μg/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 μg/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 μg/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 μg/l. To the best of our knowledge, this titer is the highest achieved by . This study lays the foundation for further engineering of for efficient 5-MTHF biosynthesis.
L-5-甲基四氢叶酸（5-MTHF）是叶酸的生物活性形式之一，被广泛用作营养保健品。然而，5-MTHF化学合成过程中的低产率和严重污染阻碍了其可持续供应。在本研究中，通过改造5-MTHF生物合成途径和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）供应来提高5-MTHF的产量，以开发一种绿色可持续的生物合成方法。具体而言，过表达关键限速酶亚甲基四氢叶酸还原酶导致细胞内5-MTHF积累，达到18μg/L。接下来，通过5-MTHF合成途径酶与亚甲基四氢叶酸还原酶的组合过表达进一步增强了5-MTHF的合成，产量提高了1.7倍。通过表达编码鸟苷三磷酸环化水解酶I来强化叶酸供应途径，使5-MTHF产量提高了2.4倍，达到72μg/L。此外，过表达6-磷酸葡萄糖脱氢酶以改善5-MTHF生物合成的氧化还原辅因子NADPH供应，这导致细胞内NADPH增加60%，5-MTHF产量增加35%（97μg/L）。为了减少副产物5-甲酰四氢叶酸的形成，过表达5-甲酰四氢叶酸环化连接酶将5-甲酰四氢叶酸转化为5,10-甲基四氢叶酸，这使5-MTHF滴度提高到132μg/L。最后，向发酵培养基中组合添加叶酸前体提高了5-MTHF产量，达到300μg/L。据我们所知，这个滴度是通过……实现的最高值。本研究为进一步改造……以实现高效5-MTHF生物合成奠定了基础。
