Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Biotechnol Bioeng. 2020 Jul;117(7):2116-2130. doi: 10.1002/bit.27332. Epub 2020 Apr 14.
5-Methyltetrahydrofolate (5-MTHF) is the major form of folate in human plasma and is the only folate form that can penetrate the blood-brain barrier. It has been widely used for the prevention and treatment of various diseases. It is mainly produced by chemical synthesis. However, the low production rate cannot meet the increasing demand. In addition, chemical synthesis is potentially detrimental to the environment. Despite various microorganisms synthetizing 5-MTHF, an efficient 5-MTHF bioproduction approach is lacking because of the tight regulation of the 5-MTHF pathway and limited metabolic flux toward the folic acid pathway. In this study, the 5-MTHF synthetic pathway in Bacillus subtilis was systematically engineered to realize 5-MTHF accumulation and further improve 5-MTHF production. Specifically, the 5-MTHF synthesis pathway with dihydrofolate (DHF) as the precursor was strengthened to shift the metabolic flux to 5-MTHF biosynthesis by replacing the native yitJ gene with Escherichia coli metF, knockout of purU, and overexpressing dfrA. The intracellular level of 5-MTHF increased 26.4-fold, reaching 271.64 µg/L. Next, the 5-MTHF precursor supply pathway was strengthened by co-overexpression of folC, pabB, folE, and yciA. This resulted in a 93.2-fold improvement of the 5-MTHF titer, which reached 960.27 µg/L. Finally, the clustered regularly interspaced short palindromic repeats interference system was used to identify key genes in the competitive and catabolic pathways for repression to further shift the metabolic flux toward 5-MTHF biosynthesis. The repression of genes thyA (existing in the purine metabolic pathway), pheA (existing in the competitive metabolic pathway), trpE (existing in the competitive metabolic pathway), and panB (existing in the pantoate synthesis pathway) significantly increased the titer of 5-MTHF. By repressing the pheA gene, the 5-MTHF titer reached 1.58 mg/L, which was 153.8-fold that of the wild-type strain of B. subtilis 168. Through medium optimization, the 5-MTHF titer reached 1.78 mg/L, which was currently the highest titer of 5-MTHF in B. subtilis. Apart from the highest titer of 5-MTHF, the highest titer of total folates including 5-MTHF, 5-FTHF, folic acid, and THF could reach 3.31 mg/L, which was 8.5-fold that in B. subtilis. To the best of our knowledge, the 5-MTHF and total folate titers reported here are the highest using a Generally regarded as safe (GRAS) bacterium as the production host. Overall, this study provides a good starting point for further metabolic engineering to achieve efficient biosynthesis of 5-MTHF by GRAS bacteria.
5-甲基四氢叶酸（5-MTHF）是人体血浆中叶酸的主要形式，也是唯一能够穿透血脑屏障的叶酸形式。它已被广泛用于预防和治疗各种疾病。它主要通过化学合成生产。然而，低产率无法满足不断增长的需求。此外，化学合成可能对环境有害。尽管有各种微生物合成 5-MTHF，但由于 5-MTHF 途径的严格调控和代谢通量向叶酸途径的限制，缺乏有效的 5-MTHF 生物生产方法。在这项研究中，通过系统工程化枯草芽孢杆菌中的 5-MTHF 合成途径来实现 5-MTHF 积累，并进一步提高 5-MTHF 的产量。具体来说，通过用大肠杆菌 metF 替换天然 yitJ 基因、敲除 purU 和过表达 dfrA，强化以二氢叶酸（DHF）为前体的 5-MTHF 合成途径，将代谢通量转向 5-MTHF 生物合成。5-MTHF 的细胞内水平增加了 26.4 倍，达到 271.64μg/L。接下来，通过共过表达 folC、pabB、folE 和 yciA 来增强 5-MTHF 前体供应途径。这使得 5-MTHF 的产量提高了 93.2 倍，达到 960.27μg/L。最后，使用成簇规则间隔短回文重复干扰系统来鉴定竞争和分解代谢途径中的关键基因，以进一步将代谢通量转向 5-MTHF 生物合成。抑制存在于嘌呤代谢途径中的 thyA 基因、存在于竞争代谢途径中的 pheA 基因、存在于竞争代谢途径中的 trpE 基因和存在于 pantothenate 合成途径中的 panB 基因显著提高了 5-MTHF 的产量。通过抑制 pheA 基因，5-MTHF 的产量达到 1.58mg/L，是枯草芽孢杆菌 168 野生型菌株的 153.8 倍。通过培养基优化，5-MTHF 的产量达到 1.78mg/L，这是目前枯草芽孢杆菌中 5-MTHF 的最高产量。除了 5-MTHF 的最高产量外，包括 5-MTHF、5-FTHF、叶酸和 THF 在内的总叶酸的最高产量可达到 3.31mg/L，是枯草芽孢杆菌的 8.5 倍。据我们所知，使用公认安全（GRAS）细菌作为生产宿主，这里报告的 5-MTHF 和总叶酸的产量是最高的。总的来说，这项研究为通过 GRAS 细菌实现 5-MTHF 的高效生物合成提供了一个良好的起点。
