Part:BBa_K4886004

Pfba-FXpk(BD)

It is a part that is responsible for expressing F/Xpk from Clostridium tyrobutyricum. with fba promotor. X-FXpk(BD) vector (7670 bp) was obtained by using the constructed recombinant plasmid Pthl-FXpk(BD) as template and X-PN-F and X-PN-R as primers. Using the genome of Clostridium butyricum as template, P-Ptkt-F and P-Ptkt-R as primers, Ptkt gene fragment (300bp) was amplified. Gibson assembly method was used to connect the Ptkt fragment to the X-FXpk(BD) linearized vector. Colony PCR (1396 bp) was performed on the transformed colonies with primers P-Ptkt-F and CX. The positive colonies with correct colony PCR were transferred, plasmid was extracted, and the recombinant plasmid was obtained after sequencing verification.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1689
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1689
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

（1）Plasmid construction

By using a recombinant plasmid pMTL-Pthl-F/Xpk(BD) as a template, and X-PN-F and X-PN-R as primers, we obtained a X-F/Xpk(BD) vector (7670 bp). Pfba fragment (300bp) was amplified from the genome template of C. acetobutylicum using P-Pfba-F and P-Pfba-R as primers, by PCR. DNA electrophoresis confirmed the lengths of the PCR products (Figure 2). Pfba fragment was ligated with X-F/Xpk(BD) vector into a pMTL-Pfba-FXpk(BD) recombinant plasmid by Gibson assembly. The plasmid was transformed into a E. coli JM109 strain. After verification by colony PCR and DNA electrophoresis (623 bp), positive colonies were transferred and expanded. Gene sequencing was used to verify that the plasmid extracted from the colonies was pMTL-Pfba-FXpk(BD).

Figure 2 Verification of Pfba (300bp) by DNA gel electrophoresis

（2）Transfection and function analysis

By using E. coli CA434 as a donor strain, pMTL-Pfba-F/Xpk(BD) was transferred to C. tyrobutyricum, notated as Ct(Pfba F/Xpk-BD). Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) (refer to BBa_K4886006) and Ct(Pthl F/Xpk-BD) (refer to BBa_K4886001) were fermented using xylose as carbon source. Fermentation experiment showed that the growth of Ct(Ptkt F/Xpk-BD) was better than that of Ct(Pthl F/Xpk-BD), and the growth of Ct(Pfba F/Xpk-BD) was worse than that of Ct(Pthl F/Xpk-BD) (Figure 3).

Figure 3 Growth performance of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD) on xylose

HPLC experiment showed that after culturing on xylose for 60.5h, the yield of butyric acid was 5.89 g/L in Ct(Ptkt F/Xpk-BD), higher than the 5.19 g/L yield in Ct(Pthl F/Xpk-BD) and the 5.21 g/L in Ct(Pfba F/Xpk-BD). Ct(Ptkt F/Xpk-BD) showed higher xylose consumption than Ct(Pthl F/Xpk-BD) and Ct(Pfba F/Xpk-BD) (Figure 4).

Figure 4 Butyric acid yield and xylose consumption of Ct(Pfba F/Xpk-BD), Ct(Ptkt F/Xpk-BD) and Ct(Pthl F/Xpk-BD)

The results implied that among Pthl, Ptkt and Pfba, Ptkt was the best promoter for F/Xpk(BD) gene to construct NOG pathway in C. tyrobutyricum to have satisfactory growth, butyric acid production and carbon conservation.