Part:BBa_K4886006
Ptkt-P/Xpk(BD)
It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Ptkt promotor. It consists of Ptkt sequence (BBa_K4886005), strong ribosomal binding site (RBS) sequence (BBa_K103015), F/Xpk sequence (BBa_K4119076) and terminator sequence (BBa_K3585002). F/Xpk is a gene that encodes phosphoketolase. Phosphoketolase is an enzyme with both the Fpk and Xpk activity. It catalyzes the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), and the conversion of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1675
Illegal XbaI site found at 618 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1675
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1675
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1675
Illegal XbaI site found at 618 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1675
Illegal XbaI site found at 618 - 1000COMPATIBLE 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). Ptkt fragment (300bp) was amplified from the genome template of C. acetobutylicum using P-Ptkt-F and P-Ptkt-R as primers, by PCR. DNA electrophoresis confirmed the lengths of the PCR products (Figure 2). Ptkt fragment was ligated with X-F/Xpk(BD) vector into a pMTL-Ptkt-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, positive colonies were transferred and expanded. Gene sequencing was used to verify that the plasmid extracted from the colonies was pMTL-Ptkt-FXpk(BD).
Figure 2 Verification of Ptkt (300bp) by DNA gel electrophoresis
(2)Transfection and function analysis
By using E. coli CA434 as a donor strain, pMTL-Ptkt-FXpk(BD) plasmid was transferred to C. tyrobutyricum, noted as Ct(Ptkt F/Xpk(BD)). C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(BD) was used as the control. C. tyrobutyricum transfected with pMTL-Pfba-F/Xpk(BD) was noted as Ct(Pfba F/Xpk(BD)). pMTL-Pthl-F/Xpk(BD) and pMTL-Pfba-F/Xpk(BD) plasmids were same as pMTL-Ptkt-FXpk(BD) except using a different promoter, Pthl or Pfba. Xylose was used as the carbon source for fermentation.
Fermentation experiment showed that the growth of Ct(Ptkt F/Xpk(BD)) was worse than that of the control and that of Ct(Pfba F/Xpk(BD)) (Figure 3).
HPLC experiment showed that after culturing on xylose for 48h, the yield of butyric acid was 2.02 g/L in Ct(Ptkt F/Xpk(BD)), lower than the 2.40 g/L yield in the control and the 2.58 g/L yield in Ct(Pfba F/Xpk(BD)) (Figure 4).
From this, it can be concluded that using Ptkt as the promoter for F/Xpk(BD) gene was not satisfactory for growth and butyric acid production in the NOG pathway of the engineered C. tyrobutyricum, compared with Pthl and Pfba.
Figure 3 Growth performance of Ct(Pfba F/Xpk(BD)) and Ct(Ptkt F/Xpk(BD)) （Control：Ct(F/Xpk-BD) with Pthl promoter）
Figure 4 Butyric acid yield and glucose consumption of Ct(Pfba F/Xpk(BD)) and Ct(Ptkt F/Xpk(BD))（Control：Ct(F/Xpk-BD) with Pthl promoter）
| None |