Part:BBa_K4885004:Design
Pthl-adhE2-Pcat1-Cat1
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
By comparing the butanol synthesis pathway of Clostridium acetobutylicum and the butyrate synthesis pathway of C. tyrobutyricum, it was found that we can introduce a single enzyme, an alcohol/aldehyde bifunctional dehydrogenase, to construct a butanol synthesis pathway in C. tyrobutyricum. By expressing adhE2 gene in C. tyrobutyricum, we can construct the butanol synthesis pathway. We used a strong transcriptional promoter, Pthl, in C. tyrobutyricum L319 for robust expression of adhE2. In C. tyrobutyricum L319, glucose is converted to pyruvate and subsequently to acetyl-CoA. Then, one part of acetyl-CoA is converted to acetate, another part is converted to ethanol, and the rest is converted into butyryl-CoA. Butyryl-CoA is respectively converted to butyrate by butyryl-CoA/acetate CoA transferase. The CoA transferase also converts acetate back to acetyl-CoA. CoA transferase reduces the acetyl-CoA to acetate flux and enhances the acetyl-CoA to butyrate flux. Therefore, the higher the expression of CoA transferase, the higher the butyrate/acetate ratio in the final product. The endogenous cat1 gene of C. tyrobutyricum L319 codes the butyryl-CoA/acetate CoA transferase. Using the cat1 gene and the native Pcat1 promoter of this gene, we overexpressed cat1 in C. tyrobutyricum, in order to suppress the acetyl-CoA conversion to acetate and indirectly enhance the metabolic flux of butyrate and butanol synthesis.
Source
Pthl sequence is from BBa_K3443002; RBS sequence is from BBa_K103015; adhE2 sequence is from BBa_K1462060; terminator sequence is from BBa_K3585002; Pcat1 promoter sequence is from BBa_K4119011. cat1 sequence is from BBa_K4119031.