Our project focuses on enhancing the production of L-5-methyltetrahydrofolate (L-5-MTHF) by engineering E. coli strains through targeted modifications. We constructed two key plasmids, pRSF-metF-folA and pET-ftfL-mtdA-fchA, each designed to express the enzymes crucial for synthesizing L-5-MTHF. The conversion process begins with folic acid, which is reduced to THF by DHFR encoded by the folA gene. THF is then converted to N¹⁰-formyl-THF by FTFL, followed by a series of enzymatic transformations involving Formyl-THF Cyclohydrolase (FCHA, fchA) and Methylene-THF Dehydrogenase (MTDA, mtdA). The final step consists of the reduction of N⁵, N¹⁰-methylene-THF to the bioactive L-5-MTHF by Methylenetetrahydrofolate Reductase (MTHFR, metF). By constructing plasmids that drive the expression of these key enzymes, we aim to improve the efficiency of this conversion process, thereby boosting L-5-MTHF production in E. coli.

Figure 1. The engineering design schematic diagram

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 strong, 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.

Figure 2. The plasmid map of pRSFDuet-metF-folA.

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.

Figure 3. The gel electrophoresis validation of metF (Left) and folA (Right) nucleic acids.

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

Figure 4. Transformation plate of pRSFDuet-metF-folA (A); Enzyme digestion verification for DH5α: pRSF-metF (B), pRSF-metF-folA (C); Sequencing results of pRSFDuet-metF-folA (D).

The pRSFDuet-metF-folA plasmid was transformed into E. coli BL21 (DE3) to evaluate the co-expression of the metF and folA 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 metF and folA proteins, particularly under induction at 37 ℃. Western Blot analysis confirmed the successful expression of all proteins, demonstrating effective co-expression.

Figure 5. Expression of metF and folA Proteins in BL21 (DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)

The enzyme digestion verification confirmed that the pRSF-metF-folA plasmid was successfully constructed, as evidenced by the expected target bands in the gel electrophoresis results. The successful amplification and ligation of the metF and folA genes demonstrate the effectiveness of the selected restriction sites and the compatibility of the pRSFDuet-1 vector for multi-gene insertion. Moving forward, the focus will be on optimizing the expression conditions for metF and folA in E. coli BL21 (DE3), with particular attention to factors such as induction temperature, IPTG concentration, and post-transformation growth conditions. Additionally, exploring different promoter strengths and ribosome binding site sequences could further enhance protein expression levels, ensuring a more efficient L-5-MTHF production pathway.

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).

Figure 6. The plasmid map of pETduet-ftfL-mtdA-fchA.

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.

Figure 7. The gel electrophoresis validation of ftfL (Left), mtdA, and fchA (Right) nucleic acids.

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

Figure 8. Transformation plate of pETDue-ftfL-mtdA-fchA (A);Enzyme digestion verification for DH5α: pETDue-ftfL F (B), pETDue-ftfL-mtdA-fchA (C);Sequencing results of pETDue-ftfL-mtdA-fchA (D).

The 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 ℃. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

Figure 9. Expression of metF, folA, mtdA, fchA, and ftfL Proteins in BL21 (DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)

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.

Figure 10: One-Step Growth Curve for BL21, pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A.

The successful co-expression of ftfL, mtdA, and fchA genes confirmed the effectiveness of the plasmid design and construction process. However, the data also suggested that the expression levels of the three proteins were not perfectly balanced, indicating room for optimization. The expression was notably more efficient at 37 ℃, which suggests that this temperature is more suitable for the co-expression of these genes.

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. 11, 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.

Figure 11. Variation of L-5-MTHF production over time.

In conclusion, we successfully increased the production of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21. Notably, the product of methionine synthase MTRR (encoded by the metH gene) in the metabolic pathway inhibits the activity of MTHFR in the L-5-MTHF synthesis pathway, which further affects the yield of L-5-MTHF. Therefore, in the future, we will continue to study the metH gene in the metabolic pathway and explore the effect of its knockdown on the whole metabolic pathway and the strain itself.

[1]Ismail S, Eljazzar S, Ganji V. 2023. Intended and Unintended Benefits of Folic Acid Fortification—A Narrative Review. Foods 12:1612.