Part:BBa_K4408008
Pthl-adhE2, Expression of adhE2 with Pthl promoter
This part is responsible for expressing adhE2 protein. The expression is controlled by a Pthl promoter with an ribosome binding site (RBS) and a Cpa fdx terminator. It consists of BBa_K3443002(Pthl), BBa_K4408001(RBS for adhE2), BBa_K1462060(adhE2), and BBa_K3585002(Cpa fdx terminator). In our program, we use this device to construct the synthesis pathways of butanol in Clostridium tyrobutyricum. By introducing adhE2，butyryl-CoA is converted to butyl aldehyde and further transformed to butanol. Pthl starts the transcription and Cpa fdx terminator ends the transcription.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
(1)Plasmid construction
We took a recombinant plasmid pMTL-Bs2 constructed previously by our research group as template, and used PCR to obtain a linearized pMTL-Pthl vector. adhE2 gene fragment was amplified from the genome of Clostridium acetobutylicum by PCR. DNA electrophoresis confirmed the lengths of the two PCR products (5461bp, and 2577bp). adhE2 gene fragment was ligated with pMTL-Pthl linearized vector into a pMTL-Pthl-adhE2 recombinant plasmid by Gibson assembly method. pMTL-Pthl-adhE2 was transformed into E. coli JM109 strain. Colony PCR and DNA electrophoresis (750 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMTL-Pthl-adhE2 by gene sequencing.
(2)Function of plasmid in C. tyrobutyricum
The plasmid pMTL-Pthl-adhE2 was transferred to C. tyrobutyricum. Protein gel electrophoresis showed that aldehyde dehydrogenase (94 kDa) encoded by adhE2 was expressed in the engineered "C. tyrobutyricum" (Figure 1). Fermentation experiment showed that adhE2 overexpression did not affect the growth of the strain (Figure 2). HPLC experiment showed that 3.0 g/L butanol and 1.0 g/L butyrate were obtained from this strain, thus realizing a de novo synthesis pathway of butanol while maintaining the native synthesis pathway of butyrate (Figure 3).
| None |