L-5-methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. The production of L-5-MTHF using microbes is gaining attention as a method for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. In this study, Escherichia coli BL21(DE3) was engineered to enhance the production of L-5-MTHF by overexpressing the intrinsic genes for dihydrofolate reductase (folA) and methylenetetrahydrofolate reductase (metF), which were cloned into the pRSFDuet-1 vector to create the pRSFDuet-metF-folA plasmid. Additionally, genes encoding formate-THF ligase (ftfL), formyl-THF cyclohydrolase (fchA), and methylene-THF dehydrogenase (mtdA) were cloned into the pETDuet-1 vector to form the pETDuet-ftfL-mtdA-fchA plasmid. By constructing these plasmids to drive the expression of key enzymes, we aim to improve the efficiency of the conversion process and thereby enhance L-5-MTHF production in E. coli.

The pRSFDuet-metF-folA plasmid was constructed by selecting and amplifying the metF and folA genes to optimize the L-5-MTHF production pathway. The pRSFDuet-1 vector was chosen due to its capability to accommodate multiple gene insertions and its robust and regulated expression under the T7 promoter. The metF gene (894 bp) and folA gene (480 bp) were amplified via PCR and inserted into the pRSFDuet-1 vector.

The metF and folA genes were successfully amplified using PCR, yielding bands of 894 bp and 480 bp, respectively. The metF gene was inserted into the pRSFDuet-1 vector by digestion with BamHI and HindIII, while the folA gene was inserted into the vector by digestion with NdeI and XhoI. The recombinant plasmid was then transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as evident from the expected band sizes in gel electrophoresis.

Upon verifying the successful amplification of the targeted plasmid, they were then transformed into E.coli DH5α. We selected colonies and sequenced them.

The connected vectors were verified by enzyme digestion, and the exact target bands were obtained respectively. The first two lanes show successful enzyme digestion verification for pRSF-metF-folA, Which indicates that the destination segment is successfully connected.

The pETDuet-ftfL-mtdA-fchA plasmid was constructed with the objective of enhancing the L-5-MTHF synthesis pathway by co-expressing the ftfL, mtdA, and fchA genes. The pETDuet-1 vector was selected for its dual-expression system, utilizing the T7 promoter to drive synchronized expression of these critical enzymes. The ftfL gene (1685 bp) was inserted first, followed by the mtdA-fchA fragment (1488 bp), ensuring that all genes were under the control of the T7 promoter for optimal co-expression in E.coli BL21(DE3).

The ftfL gene (1685 bp) and the mtdA-fchA fragment (1488 bp) were successfully amplified using PCR. The ftfL gene was inserted into the pETDuet-1 vector by digestion with BamHI and HindIII, while the mtdA-fchA fragment was inserted by digestion with NdeI and KpnI. The resulting plasmid was transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as indicated by the expected band sizes in gel electrophoresis.

To further investigate the production pathway of L-5-MTHF, we co-transformed the constructed pETduet-ftfL-fchA-mtdA recombinant plasmid, which was completed, with the pRSFduet-metF-folA recombinant plasmid into E. coli BL21 (DE3). It was verified by colony PCR to prove that strain A was constructed successfully.

The pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA plasmid was transformed into E. coli BL21(DE3) to evaluate the co-expression of the ftfL, mtdA, and fchA genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the FtfL, mtdA, and fchA proteins, particularly under induction at 37°C. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

A one-step growth curve was generated to compare the growth rates of the different strains. The control strain, BL21, initially entered a rapid growth phase, transitioning into the stationary phase after approximately 10 hours. In contrast, the strains pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A (which contains both the pRSF and pET plasmids) exhibited slower initial growth rates compared to the control strain. Notably, Strain A showed continued growth even at the 12-hour mark, indicating a prolonged exponential growth phase. This suggests that Strain A may have a higher potential for sustained growth, likely due to the combined effects of both plasmids, which enhance L-5-MTHF production and lead to a more robust metabolic capacity under the given conditions.

To further determine the actual yield of L-5-MTHF, we utilised HPLC. Firstly, we inoculated the constructed host bacteria into 100 mL of LB medium at 1% inoculum, supplemented the medium with folic acid (0.13 g/L) and sodium formate (0.013 g/L), and incubated the medium at 37℃ and 220 rpm until the OD600 value was 0.8, then added IPTG at a final concentration of 0.8 mM, and the temperature was lowered to 18℃, and the rotational speed was lowered to 110 rpm. Induction culture was carried out for 12-48h.

Subsequently, the samples were processed, 50 mL of bacterial solution was taken, centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was discarded, and resuspended to the same volume in an anaerobic chamber with Tris-HCl (pH 7.2) containing 0.1% ascorbic acid and 0.1% mercaptoethanol, sealed in an anaerobic vial, and the cells were broken by boiling for 10 min in a 100°C water bath and immediately ice-bathed. 15, 000 rpm at 4°C, 15 min after centrifugation, and the supernatant was collected, fresh mouse serum (100 μl/mL)37 was added for 3 h, and the reaction was terminated by boiling for 5 min. 000 rpm, 4°C centrifugation for 15 min and collect the supernatant, add fresh mouse serum (100 μl/mL)37 reaction for 3 h, boil for 5 min to terminate the reaction, centrifugation to take the supernatant, filtered with 0.22 μm cellulose acetate filter membrane, and loaded into the liquid-phase vial. The product measured experimentally was L-5-MTHF, and its HPLC conditions were fluorescence detector Ex=290 nm, Em=356 nm.

As can be seen from Table 1, the actual amount of L-5-MTHF produced by strain A after co-transformation was higher compared to the control BL21 strain, which indicates that the addition of folic acid ultimately produces biologically active L-5-MTHF by the enzyme expressed by strain A.

In addition, as can be seen from Fig. 8, the data measured for the control strain BL21 did not fluctuate significantly with the prolongation of the incubation time; whereas for the co-transformed strain A, the final production of active L-5-MTHF increased gradually and reached the maximum value at 48 h. Due to the oxidation of samples and depletion of samples during the experimental sampling process, the actual production of active L-5-MTHF should be slightly higher than the measured value. It is worth affirming that we successfully increased the yield of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21.