One-pot enzymatic synthesis of l-5-methyltetrahydrofolate from folic acid using enzyme cascades†

L-5-Methyltetrahydrofolate (L-5-MTHF) is an active form of folate and widely used as a nutraceutical due to its high bioavailability. Herein, we report an efficient one-pot three-enzyme cascade reaction for the production of L-5-MTHF starting from synthetic folic acid (FA). The newly-designed synthesis route was validated by enzyme screening and process optimization. The highly-active dihydrofolate reductase from Lactobacillus bulgaricus (LbuDHFR) was identified for asymmetric hydrogenation towards unnatural substrate FA, which could remarkably increase the synthetic efficiency. Dimethylsulfoniopropionate-dependent demethylase (DmdA) was successfully employed for directly converting tetrahydrofolate into L-5-MTHF using a cheap methyl donor. RcoDmdA from marine bacteria Ruegeria conchae was selected due to its high tolerance against the inhibition of the demethylated by-product. The optimal one-pot enzymatic synthesis could completely convert 34 mM of FA into 32.5 mM of L-5-MTHF with a molar conversion rate of 95.6%. No FA, dihydrofolate or tetrahydrofolate were detected in the final reaction mixture. Therefore, the new one-pot enzymatic method, circumventing the need for a transition metal catalyst, an unstable strong reductant and crystallization resolution, is proved to be simple, cost-effective, and easy to scale up for the green synthesis of L-5-MTHF.