Part:BBa_K4885002:Design
Pthl-adhE2-Dac
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3300
- 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. We propose that in C. tyrobutyricum, acetylation and deacetylation are important in reducing intermediate accumulation and controlling the carbon flow to different products such as butyrate and acetate by regulating the activities of related enzymes. The deacetylase coded by Dac plays an essential role in the deacetylation in C. tyrobutyricum. We used this native Dac gene to construct the recombinant plasmid of pMTL-Pthl-adhE2-Pcat1-Dac to overexpress adhE2 and Dac in C. tyrobutyricum. We used a strong transcriptional promoter, Pcat1, in C. tyrobutyricum L319 for robust expression of Dac. By enhancing the expression of deacetylase, we can enhance the acetylation and deacetylation interplay in the synthesis pathway of butyrate and butanol, and thus improve the efficiency of the pathway. In this way, the yields of butyrate and butanol can be increased.
Source
Pthl sequence is from BBa_K3443002
RBS sequence is from BBa_K103015
adhE2 sequence is from BBa_K1462060
Dac sequence is from BBa_K4885000
terminator sequence is from BBa_K3585002