Part:BBa_K5189006

pRSFduet-metF-folA

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 137
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 293
  Illegal BamHI site found at 106
  Illegal XhoI site found at 342
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 312
  Illegal AgeI site found at 554
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 627
  

Composite Part: BBa_K5189006 (pRSFduet-metF-folA)

Construction Design

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 1: The plasmid map of pRSFduet-metF-folA

Cultivation, Purification, and SDS-PAGE

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 using NdeI and XhoI. The recombinant plasmid was then transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, with gel electrophoresis confirming successful ligation.

Figure 2: The gel electrophoresis validation of metF (Left) and folA (Right) nucleic acids

Upon verifying the successful amplification of the targeted plasmid, they were transformed into E. coli DH5α. Selected colonies were sequenced for verification.

Figure 3: Transformation plate of pRSFDuet-metF-folA (A); Enzyme digestion verification (B), (C); Sequencing results (D)

Characterization and Measurement

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°C. Western Blot analysis confirmed the successful expression of all proteins, demonstrating effective co-expression.

Figure 4: Expression of metF and folA Proteins in BL21(DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)

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

Figure 5: Construction of co-transformed strains; A: Colony PCR validation for pRSFduet-metF-folA; B: Colony PCR validation for pETduet-ftfL-mtdA-fchA; C: Colony A plate diagram

Functional Test

1. One-Step Growth Curve Analysis

A one-step growth curve was generated to compare the growth rates of different strains. The control strain, BL21, exhibited rapid growth, transitioning into the stationary phase after about 10 hours. In contrast, strains pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A (containing both the pRSFDuet-metF-folA and pETduet-ftfL-mtdA-fchA plasmids) showed slower initial growth rates but continued growing past the 12-hour mark. This suggests that Strain A may have a higher potential for sustained growth due to the combined effects of both plasmids enhancing L-5-MTHF production.

Figure 6: One-Step Growth Curve for BL21, pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A

2. HPLC Assay for L-5-MTHF Yield

To determine the actual yield of L-5-MTHF, we utilized HPLC. The constructed host bacteria were inoculated into LB medium supplemented with folic acid and sodium formate, and incubated at 37°C. After inducing protein expression with IPTG, L-5-MTHF concentrations were measured at different time intervals using HPLC.

The measured results show that strain A, after co-transformation, produced higher L-5-MTHF compared to the control BL21 strain. The data indicated that the addition of folic acid and the expression of the enzyme in Strain A led to a higher yield of biologically active L-5-MTHF.

Table 1. L-5-MTHF concentration of BL21 and Strains A

Time	L-5-MTHF concentration of BL21 (16℃)	L-5-MTHF concentration of BL21 (37℃)	L-5-MTHF concentration of Strains A (16℃)	L-5-MTHF concentration of Strains A (37℃)
0h	0.308	0.268	0.451	0.400
2h	0.322	0.222	0.605	0.488
4h	0.315	0.250	0.697	0.550
10h	0.328	0.234	0.719	0.608
22h	0.375	0.276	0.836	0.714
32h	0.304	0.228	0.946	0.821
48h	0.331	0.256	1.498	0.926

The table shows that L-5-MTHF concentration in Strain A was significantly higher than the control BL21 strain at all time points, indicating that the co-expression of the plasmids enhanced L-5-MTHF production.

Figure 7: Variation of L-5-MTHF production over time

The data in Figure 7 shows that the control strain BL21 did not experience significant fluctuations in L-5-MTHF production over time. In contrast, Strain A saw a gradual increase in L-5-MTHF production, reaching its maximum value at 48 hours. Due to oxidation and sample depletion during the experimental process, the actual production of active L-5-MTHF may have been slightly higher than the measured values.

Summary

In conclusion, we successfully increased the production of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21. The co-expression of the metF, folA, ftfL, mtdA, and fchA genes enhanced the yield of biologically active L-5-MTHF. 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, further affecting the yield of L-5-MTHF. Future studies will explore the impact of knocking down the metH gene on the entire metabolic pathway and the strain's growth characteristics.

References

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