Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Engineering of Biosynthesis Pathway and NADPH Supply for Improved L-5-Methyltetrahydrofolate Production by Lactococcus lactis

  • Lu, Chuanchuan (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Liu, Yanfeng (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Li, Jianghua (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Liu, Long (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Du, Guocheng (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University)
  • Received : 2019.11.04
  • Accepted : 2019.12.17
  • Published : 2021.01.28
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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 ㎍/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 ㎍/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 ㎍/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 ㎍/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 ㎍/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.

Keywords

References

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