Engineering of Biosynthesis Pathway and NADPH Supply for Improved L-5-Methyltetrahydrofolate Production by Lactococcus lactis

Abstract

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 μg/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 μg/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 μg/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 μg/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 μg/l. To the best of our knowledge, this titer is the highest achieved by L. lactis . This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

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

“…Thus, folic acid and sodium formate were added to the culture medium to explore whether the engineered strains were able to assimilate exogenous folic acid and formate to further improve the production of L ‐5‐MTHF. In addition, the DHFR and MTHFD enzymes in the engineered strains are specific to NADPH (D'Ari & Rabinowitz, 1991 ; Morrison & Stone, 1988 ), and intracellular glucose can provide additional NADPH through glucose‐6‐phosphate dehydrogenase (Lu et al, 2019 ). Therefore, the effects of supplementation with glucose on the synthesis of L ‐5‐MTHF were also studied.…”

“…This procedure is complicated and requires toxic chemicals and strong reductants, which may cause production safety problems and environmental pollution. Microorganisms can naturally synthesize the bioactive l ‐5‐MTHF (Lu et al, 2019 ; Shane & Stokstad, 1977 ). The production of l ‐5‐MTHF using microorganisms has recently been considered as an environmentally friendly and sustainable method although the yield is not high.…”

“…As a widely used lactic acid bacteria, Lactococcus lactis is favourable for producing L ‐5‐MTHF. By strengthening the supply of folate and NADPH via overexpression of some key enzymes such as glucose‐6‐phosphate dehydrogenase, the production of L ‐5‐MTHF in L. lactis was successfully improved (Lu et al, 2019 ). Escherichia coli has also been receiving much attention because of its clear genetic background, efficient genetic operation, simple culture conditions and economic large‐scale fermentation.…”

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

“…1 In Lactococcus lactis, by reinforcing 5-MTHF biosynthesis and improving NADPH supply, 5-MTHF production reached 300 μg/L. 18 Bacillus subtilis is a Generally Regarded As Safe (GRAS) bacterium that has long been used for producing industrial enzymes and nutraceuticals, 19,20 which can be promisingly used for efficient 5-MTHF bioproduction. In our previous study, strengthening the synthetic pathway from dihydrofolate (DHF) to 5-MTHF and repressing the branched pathway by the clustered regularly interspaced short palindromic repeat interference (CRISPRi) system led to 1.78 mg/L of 5-MTHF production.…”

“…By expressing the AgFOL gene and knocking out the folyl-poly gamma-glutamate synthetase (FPGS) encoding gene, the total folate production reached 6.6 mg/L with approximately 1.2 mg/L of 5-MTHF . In Lactococcus lactis , by reinforcing 5-MTHF biosynthesis and improving NADPH supply, 5-MTHF production reached 300 μg/L , which can be promisingly used for efficient 5-MTHF bioproduction.…”