Part:BBa_K4886006
Ptkt-P/Xpk(BD)
It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824, notated as F/Xpk(BD), with Ptkt promotor. It consists of Ptkt sequence (BBa_K4886005), 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. tyrobutyricum 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-F/Xpk(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-F/Xpk(BD).
(2)Transfection and function analysis
By using E. coli CA434 as a donor strain, pMTL-Ptkt-F/Xpk(BD) plasmid was transferred to C. tyrobutyricum, notated as Ct(Ptkt F/Xpk-BD). Ct(Pfba F/Xpk-BD) (refer to BBa_K4886004 ), Ct(Ptkt F/Xpk-BD) 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). 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). 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.
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