Part:BBa_K4886001

Pthl-F/Xpk(BD)

It is the part which is responsible for the expression of F/Xpk from Clostridium acetobutylicum ATCC824 with Pthl promotor. It consists of Pthl sequence (BBa_K3443002), 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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1986
  Illegal XbaI site found at 929
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1986
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1986
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1986
  Illegal XbaI site found at 929
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1986
  Illegal XbaI site found at 929
  
• 1000
  COMPATIBLE WITH RFC[1000]

results

(1)Plasmid construction

We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Clostridium acetobutylicum ATCC824 genome as a template to amplify the Phosphoketolase gene [FXpk(BD)] fragment (2391bp). The vector and fragment were confirmed by gel electrophoresis (Figure 1 and 2). The FXpk(BD) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR (1000 bp) for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-FXpk(BD) obtained was confirmed by gene sequencing.

Figure 1 Gel electrophoresis of FXpk gene from Clostridium acetobutylicum ATCC824 (2391 bp)

Figure 2 Gel electrophoresis of X-pMTL-Pthl linear vector (5461bp)

(2)Transfection of C. tyrobutyricum and its growth By using E. coli CA434 as a donor strain, pMTL-Pthl-F/Xpk(QS) plasmid and pMTL-Pthl-F/Xpk(BD) plasmid were transferred to C. tyrobutyricum, noted as Ct(F/Xpk-QS) and Ct(F/Xpk-BD), respectively. Refer to BBa_K4886002 for the construction of pMTL-Pthl-F/Xpk(QS). The native C. tyrobutyricum was the control. Fermentation experiment showed that the growth of Ct(F/Xpk-BD) is better than that of Ct(F/Xpk-QS) and that of the control (Figure 3). Figure 3 Growth comparison of Ct(F/Xpk-QS) and Ct(F/Xpk-BD) (3)Product yield of the transfected strain Butyric acid and acetic acid are the metabolites of C. tyrobutyricum and downstream products of the EMP pathway and the NOG pathway. Butyric acid is an important industrial product and contains two more carbon atoms than acetic acid per molecule. The results of high-performance liquid chromatography (HPLC) illustrated that Ct(FXpk-BD) had higher yield of butyric acid and lower yield of acetic acid than the control (Table 1). Ct(FXpk-BD) had a higher selectivity towards butyric acid, compared to the control, indicating an increased carbon atom economy in the engineered strain.

Table 1 HPLC results for products from Ct(FXpk-BD) and control strain