Difference between revisions of "Part:BBa K4886007"

Latest revision as of 14:58, 11 October 2023

F/Xpk(ASR)

This part is an ancestral F/Xpk gene predicted by ancestral sequence reconstruction (ASR), based on F/Xpk 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 is able to catalyze the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), as well that of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE 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
12
INCOMPATIBLE 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
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE 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
25
INCOMPATIBLE 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
1000
COMPATIBLE WITH RFC[1000]

Experiments and results

1.Ancestral sequence reconstruction (ASR) method

FireProt-ASR (https://loschmidt.chemi.muni.cz/fireprotasr/) was used by Mr. 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).

Figure 1 pMTL-Pthl-F/Xpk(ASR) recombinant plasmid construction

Figure 2 Genetic circuit of pMTL-Pthl-F/Xpk(ASR)

Table 1 Primer sequences

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

Figure 3 Growth comparison of C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(ASR) and that transfected with pMTL-Pthl-F/Xpk(BD)

Table 2 Product yields of C. tyrobutyricum transfected with pMTL-Pthl-F/Xpk(ASR) and that transfected with pMTL-Pthl-F/Xpk(BD) after fermentation for 42 hours

@@ Line 14: / Line 14: @@
 ==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).
+FireProt-ASR (https://loschmidt.chemi.muni.cz/fireprotasr/) was used by Mr. 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===