High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and Transcriptomics-Guided Global Metabolic Regulation,Journal of Agricultural and Food Chemistry

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High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and Transcriptomics-Guided Global Metabolic Regulation
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-05-06 , DOI: 10.1021/acs.jafc.2c01252
Han Yang _{1,

2,

3,

4} , Jinning Yang _{1,

2,

3,

4} , Cheng Liu _{1,

2,

3,

4} , Xueqin Lv _{1,

2,

3,

4} , Long Liu _{1,

2,

3,

4} , Jianghua Li _{1,

2,

3,

4} , Guocheng Du _{1,

2,

3,

4} , Jian Chen _{1,

2,

3,

4,

5} , Yanfeng Liu _{1,

2,

3,

4,

5}

Affiliation

5-Methyltetrahydrofolate (5-MTHF) is the predominant folate form in human plasma, which has been widely used as a nutraceutical. However, the microbial synthesis of 5-MTHF is currently inefficient, limiting green and sustainable 5-MTHF production. In this study, the Generally Regarded As Safe (GRAS) microorganism Bacillus subtilis was engineered as the 5-MTHF production host. Three precursor supply modules were first optimized by modular engineering for strengthening the supply of guanosine-5-triphosphate (GTP) and p-aminobenzoic acid (pABA). Next, the impact of genome-wide gene expression on 5-MTHF biosynthesis was evaluated using transcriptome analyses, which identified key genes for 5-MTHF production. The effects of potential genes on 5-MTHF synthesis were verified by observing the genes’ up-regulated by strong promoter P₅₆₆ and those down-regulated by inhibition through the clustered regularly interspaced short palindromic repeat interference (CRISPRi). Finally, a key gene for improved 5-MTHF biosynthesis, comGC, was integrated into the genome of modular engineered strain B89 for its overexpression and facilitating efficient 5-MTHF synthesis, reaching 3.41 ± 0.10 mg/L with a productivity of 0.21 mg/L/h, which was the highest level achieved by microbial synthesis. The engineered 5-MTHF-producing B. subtilis developed in this work lays the foundation of further enhancing 5-MTHF production by microbial fermentation, which can be used for isolation and purification of 5-MTHF as food and nutraceutical ingredients.

中文翻译：

结合模块化工程和转录组学引导的全球代谢调控，在枯草芽孢杆菌中进行高水平 5-甲基四氢叶酸生物生产

5-甲基四氢叶酸 (5-MTHF) 是人体血浆中的主要叶酸形式，已被广泛用作营养保健品。然而，目前 5-MTHF 的微生物合成效率低下，限制了绿色和可持续的 5-MTHF 生产。在这项研究中，一般认为安全 (GRAS) 微生物枯草芽孢杆菌被设计为 5-MTHF 生产宿主。三个前体供应模块首次通过模块化工程优化，以加强鸟苷-5-三磷酸（GTP）和对氨基苯甲酸（pABA)。接下来，使用转录组分析评估全基因组基因表达对 5-MTHF 生物合成的影响，该分析确定了 5-MTHF 生产的关键基因。潜在基因对 5-MTHF 合成的影响通过观察基因被强启动子 P ₅₆₆上调和通过成簇规则间隔短回文重复干扰 (CRISPRi) 抑制而下调的基因来验证。最后，将改进 5-MTHF 生物合成的关键基因comGC整合到模块化工程菌株 B89 的基因组中，使其过表达并促进高效的 5-MTHF 合成，达到 3.41 ± 0.10 mg/L，产率为 0.21 mg/L /h，这是微生物合成所达到的最高水平。工程化的 5-MTHF 生产本工作开发的枯草芽孢杆菌为进一步提高微生物发酵5-MTHF产量奠定了基础，可用于5-MTHF作为食品和保健品成分的分离纯化。

更新日期：2022-05-06

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