Part:BBa_K5189007

pETduet-ftfL-mtdA-fchA

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 149
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 305
  Illegal BglII site found at 7939
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 324
  Illegal NgoMIV site found at 671
  Illegal NgoMIV site found at 5348
  Illegal NgoMIV site found at 6321
  Illegal NgoMIV site found at 6843
  Illegal NgoMIV site found at 6984
  Illegal NgoMIV site found at 7043
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1256
  Illegal SapI.rc site found at 2916
  

Composite Part: BBa_K5189007 (pETduet-ftfL-mtdA-fchA)

Construction Design

The pETduet-ftfL-mtdA-fchA plasmid enhanced 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 1: The plasmid map of pETduet-ftfL-mtdA-fchA

Experimental Approach

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 2: The gel electrophoresis validation of ftfL (Left), mtdA, and fchA (Right) nucleic acids

Upon verifying the successful amplification of the targeted plasmid, the transformed colonies were selected and sequenced for verification.

Figure 3: Transformation plate of pETDue-ftfL-mtdA-fchA (A); Enzyme digestion verification for DH5α: pETDue-ftfL F (B), pETDue-ftfL-mtdA-fchA (C); Sequencing results (D)

Characterization and Measurement

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

Figure 4: Expression of ftfL, mtdA, fchA 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, along with the pRSFduet-metF-folA recombinant plasmid into E. coli BL21 (DE3). The colony PCR verified that Strain A was successfully constructed.

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 approximately 10 hours. In contrast, the 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.