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

Abstract

L‐5‐Methyltetrahydrofolate (L‐5‐MTHF) is the only biologically active form of folate in the human body. Production of L‐5‐MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L‐5‐MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L‐5‐MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene‐THF) reductase, introducing the genes encoding formate‐THF ligase, formyl‐THF cyclohydrolase and methylene‐THF dehydrogenase from the one‐carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L‐5‐MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT‐PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 μg g−1 of dry cell weight for L‐5‐MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L‐5‐MTHF in E. coli.

Improving Microbial Cell Factory Performance by Engineering SAM Availability

“… − Wang et al developed an additional 5-MTHF synthetic pathway in E. coli by introducing 3 exogenous genes (encoding formate-THF ligase, formyl-THF cyclohydrolase, and methylene-THF dehydrogenase respectively) from the C1 utilizing Methylobacterium extorquens or Clostridium autoethanogenum . In conclusion, E.…”

“…75−78 Wang et al developed an additional 5-MTHF synthetic pathway in E. coli by introducing 3 exogenous genes (encoding formate-THF ligase, formyl-THF cyclohydrolase, and methylene-THF dehydrogenase respectively) from the C1 utilizing Methylobacterium extorquens or Clostridium autoethanogenum. 79 In conclusion, E. coli utilizes L-serine as the ultimate methyl donor, while some C1 utilizing organisms can utilize formate as the ultimate methyl donor (Figure 2). Because formate can be efficiently produced from waste CO 2 by electrochemical reduction, 80 combining the methylation with electrochemistry should be a promising approach to efficient and eco-friendly SdNP biomanufacturing.…”

Continuous Flow Synthesis of 5-Methyltetrahydrofolate from Folic Acid

“…6−10 In view of this, the efficient synthesis of 5-MTHF has always been the primary task. Though many efforts have been made, the synthesis of 5-MTHF with FA as the starting material has been found to be the only reliable route in industry 11 because folic acid is easily obtainable commercially at economically convenient prices. 12 In fact, conversion of FA to 5-MTHF was already found several decades ago, but improvement on this process has never been stopped since then (Scheme 1).…”

“…Therefore, 5-MTHF has become a common focus of attention in the food and biomedical fields because of its remarkable efficacy and low side effects. − In view of this, the efficient synthesis of 5-MTHF has always been the primary task. Though many efforts have been made, the synthesis of 5-MTHF with FA as the starting material has been found to be the only reliable route in industry because folic acid is easily obtainable commercially at economically convenient prices …”

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

“…To find alternative sources of the formyl group, we selected the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 (Uniprot: Q83WS0, encoded by the f hs gene), which had been reported in the literature. 16 This enzyme can use formate salts as substrates to catalyze the combination of the formyl group with THF, forming 10-formyl-THF. During subsequent fermentation, we added sodium formate at double the concentration (80 mg/L), achieving a dual function for sodium formate.…”