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

Abstract

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 P 566 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.

Biosynthesis of L‐5‐methyltetrahydrofolate by genetically engineered Escherichia coli

“…The highest yield of 527.84 μg g −1 (1.24 mg L −1 ) was obtained for the engineered strain of BL21( ΔmetH )‐C1T after preliminary optimization, which was about 11.8 folds of that of the original strain. Although the yield remained lower than the reported maximum titre of L ‐5‐MTHF in Bacillus subtilis (3.41 mg L −1 ) (Yang et al, 2022 ), the production reached a relatively high level in the engineered strains when using E. coli as the host. But the current level is not high enough for large‐scale industrial production, the metabolic engineering strategies such as fine‐tuning the enzymes associated with the C1 transfer pathway from M. extorquens AM1 or the Wood–Ljungdahl pathway from C. autoethanogenum are needed to further improve the production of L ‐5‐MTHF in E. coli .…”

“…Increased production of L ‐5‐MTHF in Bacillus subtilis has been achieved by facilitating the synthesis of direct precursors of the dihydrofolate and repressing related competitive and catabolic pathways (Yang et al, 2020 ). The same group recently further improved the yield of L ‐5‐MTHF through modular engineering and global regulation of gene expression involved in the synthesis of L ‐5‐MTHF (Yang et al, 2022 ). As a widely used lactic acid bacteria, Lactococcus lactis is favourable for producing L ‐5‐MTHF.…”

Improving Microbial Cell Factory Performance by Engineering SAM Availability

“…coli and S. cerevisiae, other microbial chassis, such as Bacillus subtilis, , Lactococcus lactis, and Ashbya gossypii, have also been engineered for 5-MTHF accumulation. However, because of the long synthetic pathway and tight regulation, the titers are not high (ranging from 0.30–3.41 mg/L).…”

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

“…14 As a non-native producer of folate, Bacillus subtilis achieved high 5-MTHF levels, peaking at 3.41 mg/L, by integrating modular engineering with metabolic regulation guided by transcriptomic analysis, notably through overexpressing the essential comGC gene. 15 Similarly, Escherichia coli emerged as a promising host for 5-MTHF production due to its well-defined genetic background and comprehensive gene editing systems. By overexpressing intrinsic genes and introducing exogenous genes, the 5-MTHF output was significantly increased to 527.84 μg/g of dry cell weight, approximately 11.8-fold higher than the WT strain.…”

“…Lactococcus lactis, also a natural producer of folate, achieved a 5-MTHF yield of 300 μg/L by enhancing precursor supply and inhibiting the formation of byproducts . As a non-native producer of folate, Bacillus subtilis achieved high 5-MTHF levels, peaking at 3.41 mg/L, by integrating modular engineering with metabolic regulation guided by transcriptomic analysis, notably through overexpressing the essential comGC gene . Similarly, Escherichia coli emerged as a promising host for 5-MTHF production due to its well-defined genetic background and comprehensive gene editing systems.…”