Part:BBa_K4886007
F/Xpk(ASR)
This part is an ancestral F/Xpk gene predicted by ancestral sequence reconstruction (ASR), based on FXpk sequence (BBa_K4119076) derived from Clostridium acetobutylicum ATCC824. F/Xpk encodes phosphoketolase which is an enzyme with both the Fpk and Xpk activity. The enzyme 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 PstI site found at 779
Illegal PstI site found at 1334
Illegal PstI site found at 1928
Illegal PstI site found at 1964 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 779
Illegal PstI site found at 1334
Illegal PstI site found at 1928
Illegal PstI site found at 1964 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 779
Illegal PstI site found at 1334
Illegal PstI site found at 1928
Illegal PstI site found at 1964 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 779
Illegal PstI site found at 1334
Illegal PstI site found at 1928
Illegal PstI site found at 1964 - 1000COMPATIBLE WITH RFC[1000]
Experiments and results
1.Ancestral sequence reconstruction (ASR) method
FireProt-ASR (https://loschmidt.chemi.muni.cz/fireprotasr/) was used by Ms. Yang to carry out ASR. The phosphoketolase (F/Xpk) sequence (BBa_K4119076) derived from Clostridium acetobutylicum was used as the input sequence. No essential residues were selected. Percent sequence identity was set to 30%-70%. Clustering identity was set to 0.9. Evolutionary model was set to WAG. RAxML (Randomized Axelerated Maximum Likelihood) was chosen as the phylogenetic tree inference tool. Bootstraps were set to 500. The ancestral sequence of phosphoketolase (F/Xpk) predicted by ASR was named F/Xpk(ASR).
2.Plasmid construction
On the basis of plasmid pMTL-Pthl-F/Xpk(BD), F/Xpk(BD) fragment was replaced with F/Xpk(ASR). Using pMTL-Pthl-F/Xpk(BD) as the template and X-pMTL-F and X-pMTL-R as the primers, X-pMTL-Pthl linearized vector (5461 bp) was amplified. Using PUC57 vector plasmid as the template and P-F/Xpk(ASR)-F and P-F/Xpk(ASR)-R as the primers, F/Xpk(ASR) fragment (2436 bp) was amplified. The F/Xpk (ASR) gene fragment and the X-pMTL-Pthl linearized vector were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies using CX-FXpk-F-1 and CX-FXpk(BD)-R JP750 as the primers (957 bp). The positive colonies were transferred and the plasmid was extracted. After gene sequencing verification, the recombinant plasmid was obtained: pMTL-Pthl-F/Xpk(ASR).
3.Growth and fermentation performance of C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(ASR)
By using E. coli CA434 as a donor strain, pMTL-Pthl-F/Xpk(ASR) plasmid was transferred to C. tyrobutyricum, notated as Ct(Pthl F/Xpk-ASR). C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(BD) plasmid was used as the control, notated as Ct(Pthl F/Xpk-BD). The two engineered bacterias were then put into fermentation experiments to test their performance.
Our experiment showed that under the same experimental conditions，Ct(Pthl F/Xpk-ASR) had slightly better growth than Ct(Pthl F/Xpk-BD) (Figure 3). After fermentation for 42 hours, Ct(Pthl F/Xpk-ASR) produced more butyrate than Ct(Pthl F/Xpk-BD), with the average yield increasing from 3.52 g/L to 4.22 g/L by 20% (Table 2), which means that the metabolic flux of the NOG pathway was further increased by the use of F/Xpk(ASR) compared to Ct(Pthl F/Xpk-BD). At the same time, we also found that Ct(Pthl F/Xpk-ASR) reduced acetate synthesis. The results above indicate that F/Xpk(ASR) reconstructed using ASR has better carbon conservation than those screened in previous experiments
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