Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

Abstract

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.

High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and Transcriptomics-Guided Global Metabolic Regulation

“…38 This study assessed the impact of the global metabolic network on 5-MTHF through transcriptome analysis results and identified relevant key genes. Therefore, we selected wild-type B. subtilis 168 (CK), 5-MTHF low-producing strain A7 obtained from previous studies (0.89 mg/L of 5-MTHF 21 ), and 5-MTHF highproducing strain A9 (1.72 ± 0.04 mg/L of 5-MTHF) for transcriptional analysis. The results of the analysis were screened according to the standard of difference significance (gene expression changed more than 2-fold and q-value (adjusted p-value) ≤ 0.05) and expression fold changes of gene up-and down-regulation (Supplementary Figure S2A).…”

“…Strain A9 (B. subtilis derivate), constructed in a previous study, was used as starting host strain. 21 E. coli JM109 was used as host for gene cloning. Plasmids p7s6P1, p7s6P6, p7s6P10, p7c6P1, p7c6P6, and p7c6P10 were previously constructed based on plasmid p7s6.…”

“…Lastly, the extracts were boiled in a water bath for 15 min, the supernatant was retained, the precipitate was discarded, and the supernatant was filtered with a 0.22 μm sterile filter. 1,21 Quantification of Intracellular 5-MTHF. 5-MTHF was detected by high-performance liquid chromatography (HPLC) (1200 series fluorescence detector; Agilent, Santa Clara, CA, USA).…”

“…In our previous study, strengthening the synthetic pathway from dihydrofolate (DHF) to 5-MTHF and repressing the branched pathway by the clustered regularly interspaced short palindromic repeat interference (CRISPRi) system led to 1.78 mg/L of 5-MTHF production. 21 Because of the complex and lengthy biological pathways related to 5-MTHF biosynthesis, the synthesis of 5-MTHF using glucose as a substrate requires more than 50 steps in B. subtilis. As a result, it is challenging to fine-tune the 5-MTHF pathway and balance the precursor supply.…”

Bacillus subtilis BS-15 Effectively Improves Plantaricin Production and the Regulatory Biosynthesis in Lactiplantibacillus plantarum RX-8

“…The upregulation of MurB, MurC, and MurD at protein level suggested that there were more PG in PC than in NC, as these proteins are involved in the synthesis of PG precursors (Real and Henriques, 2006;Vasudevan et al, 2007). The GCS catalyzed the oxidative decarboxylation of glycine and was dependent on tetrahydrofolate (THF) biosynthesis (Yang et al, 2020). It is helpful to obtain energy and 5,10methylene-tetrahydrofolate, a C1 unit that is necessary for the production of serine, thymidine, and purines (Kikuchi et al, 2008).…”