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

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.

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.

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.