Part:BBa_K4885006:Design
Pthl-adhE2-bcd-crt
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3367
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4546
Illegal SapI.rc site found at 3496
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 acetyl-CoA which is then mainly converted to butyryl-CoA by the following enzymes: thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), and butyryl-CoA dehydrogenase (bcd). Butyryl-CoA is subsequently converted to butyrate in the native strain. In the preliminary experiment, we discovered that the yield of butyrate and butanol increased efficiently through the overexpression of bcd and crt. Therefore, crotonase and butyryl-CoA dehydrogenase coded by bcd and crt are considered as rate-limiting enzymes. Here, we used the endogenous bcd and crt genes and Pthl promoter from C. tyrobutyricum L319 to construct the recombinant plasmid of pMTL-adhE2-bcd-crt to overexpress adhE2, crt and bcd in C. tyrobutyricum to enhance the butyrate and butanol synthesis pathway.
Source
Pthl sequence is from BBa-K4408008.
RBS is from BBa_K4408001.
adhE2 sequence is from BBa_K1462060.
bcd sequence is from BBa_K4119027.
crt sequence is from BBa_K4119028.
Cpa fdx terminator sequence is from BBa_K3585002.