Part:BBa_K4886004:Design

Pfba-FXpk(BD)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1689
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1689
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1689
  Illegal XbaI site found at 632
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Our project aims to reduce the carbon emission during the fermentation of Clostridium tyrobutyricum (C. tyrobutyricum) by constructing a NOG pathway to integrate with the native EMP pathway of the strain. The NOG pathway is an artificial pathway and functions as a fructose-6-phosphate (F6P) shunt. Through carbon rearrangement, this pathway can convert 1mol of F6P to 3mol of AcP without any loss of carbon. By comparison, we found that most of the key enzymes in the NOG pathway natively exist in C. tyrobutyricum and the functions of the absent key enzymes can be carried out by phosphoketolases (Figure 1). Therefore, in order to construct the NOG pathway in C. tyrobutyricum L319, we introduced phosphoketolase (F/Xpk) gene into the strain. Pthl promoter is a strong transcriptional promoter in C. tyrobutyricum L319. We used this promoter to ensure a robust expression in the strain.

Figure 1 NOG pathway and related enzyme genes (red arrows indicate lack of key enzymes in Clostridium tyrobutyricum)

Source

The Pfba promoter sequence is from BBa_K4886003. The strong RBS sequence is from BBa_K103015. The F/Xpk sequence is from BBa_K4119076. The terminator sequence is from BBa_K3585002.