Improved methyl supply for 5-methyltetrahydrofolate production in E. coli through a novel C1 transfer pathway

BACKGROUND: L-5-Methyltetrahydrofolate (5-MTHF) is the sole biologically active form of folate present in human blood and serves as an essential nutritional supplement. While microbial biosynthesis of 5-MTHF offers a sustainable alternative to chemical synthesis, its low yield limits industrial potential. RESULTS: In this study, strategies for improving the methyl supply combined with engineering the tetrahydrofolate (THF) synthetic pathway were employed in E. coli to increase 5-MTHF production. First, a new exogenous C1 pathway was introduced to improve the intracellular methyl supply through acetyl-CoA breakdown. High expression of key rate-limiting genes folE, folP and purU enhanced metabolic flux of THF pathway, resulting in a 5-MTHF titer of 1.075 mg/L during shake-flask fermentation. A subsequent increase in 5-MTHF production was achieved by knocking out the metE gene, which is involved in the consumption of 5-MTHF. The best engineered strain, M3012, produced 8.2 mg/L 5-MTHF in a 5 L bioreactor via fed-batch fermentation, which presented the highest 5-MTHF titer to date. CONCLUSION: We successfully engineered E. coli by introducing a novel exogenous C1 metabolic pathway to augment the methyl donor pool essential for the biosynthesis of 5-MTHF. Further metabolic optimizations, including the enhancement of the THF precursor flux and the elimination of competing degradation pathways, developed a recombinant strain with significantly increased yield, which paves the way for industrial production of 5-MTHF.