L-5-Methyltetrahydrofolate (L-5-MTHF) is the primary form of folate in serum and the only biologically active form in humans. It is also the only folate capable of crossing the blood-brain barrier. As the bioactive form of folic acid, L-5-MTHF is crucial for nucleic acid synthesis, cell growth, tissue repair, and embryonic development (1-4). Humans are unable to synthesize folic acid and must obtain it from dietary sources. In the body, folic acid is reduced to tetrahydrofolate, the coenzyme for one-carbon transferase, through the action of dihydrofolate reductase. Tetrahydrofolate then binds to a one-carbon unit and, via a series of enzymatic reactions, is converted into the biologically active form L-5-MTHF (5). The compound can be commercially supplied to individuals with impaired folic acid metabolism. However, current commercial production primarily depends on chemical synthesis (6-9). This presents challenges such as limited supply, safety concerns, and environmental risks (10).

E. coli BL21(DE3) was selected as the host organism for our project because of its well-characterized metabolic pathways and ease of cultivation (11). We carefully analyzed and engineered a synthetic pathway for L-5-methyltetrahydrofolate (L-5-MTHF) in E. coli BL21(DE3). To begin, we overexpressed the native genes encoding dihydrofolate reductase (folA) and methylenetetrahydrofolate reductase (metF), which were cloned into the pRSFDuet-1 vector, generating the pRSFDuet-metF-folA plasmid. This plasmid was subsequently transformed into competent E. coli BL21(DE3) cells.

To enhance the production of L-5-MTHF, we selected enzymes from the C1 transfer pathway of Methylobacterium extorquens AM1. The genes encoding formate-THF ligase (ftfL), formyl-THF cyclohydrolase (fchA), and methylene-THF dehydrogenase (mtdA) were cloned into the pETDuet-1 vector, creating the pETduet-ftfL-mtdA-fchA plasmid. This plasmid was then introduced into E. coli BL21(DE3). The resulting plasmid was co-transformed into an E. coli host that already contained pRSFDuet-metF-folA.

Ultimately, we developed three engineered strains for the production of L-5-MTHF. The growth curves of these strains were consistent with those of the original E. coli BL21(DE3) strain, without the addition of IPTG, folic acid, sodium formate, or glucose. The production of L-5-MTHF by all engineered strains was quantified using HPLC under optimized conditions, and their performance was subsequently analyzed and compared.

Figure. 1 Construction of an additional pathway for synthesising L-5-MTHF in E. coli.

In this study, we aimed to develop a synthetic biology-based production system for active folate (L-5-MTHF) and enhance its production in E. coli through pathway engineering. We optimized several conditions, including induction time, temperature, cell density, and IPTG concentration, while also supplementing the culture with exogenous substances such as folic acid, sodium formate, and glucose. This approach led to the highest yield of dry cell weight for L-5-MTHF. This research paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli, making the process more efficient, cost-effective, environmentally sustainable, and safer for large-scale industrial production.

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