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.

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Currently, the primary source of ribonucleic acids (RNAs), which is used as a flavor enhancer and nutritional supplement in the food manufacturing and processing industries, for large-scale industrial production is yeast, where the challenge is to optimize the cellular RNA content. Here, we developed and screened yeast strains yielding abundant RNAs via various methods. The novel Saccharomyces cerevisiae strain H1 with a 45.1% higher cellular RNA content than its FX-2 parent was successfully generated. Comparative transcriptomic analysis elucidated the molecular mechanisms underlying RNA accumulation in H1. Upregulation of genes encoding the hexose monophosphate and sulfur-containing amino acid biosynthesis pathways promoted RNA accumulation in the yeast, particularly in the presence of glucose as the sole carbon source. Feeding methionine into the bioreactor resulted in 145.2 mg/g dry cell weight and 9.6 g/L of cellular RNA content, which is the highest volumetric productivity of RNAs achieved in S. cerevisiae. This strategy of breeding S. cerevisiae strain with a higher capacity of accumulating abundant RNAs did not employ any genetic modification and thus will be favored by the food industry.