Part:BBa_K4408009
Pthl-adhE2-thl-hbd, Expression of adhE2, thl and hbd with Pthl promoter
This part is responsible for expressing adhE2, thl and hbd proteins. The expression of these proteins are simultaneously controlled by a Pthl promoter with a ribosome binding site (RBS) and a Cpa fdx terminator. It consists of BBa_K3443002(Pthl), BBa_K103015(RBS1), BBa_K1462060(adhE2), BBa_K3443003 (thl), BBa_M1350 (hbd) and BBa_K3585002(Cpa fdx terminator). In our program, we use this device to construct and optimize the synthesis pathways of butanol and butyrate in Clostridium tyrobutyricum. By introducing adhE2，butyryl-CoA is converted to butyl aldehyde and further transformed to butanol. Enzyme encoded by thl catalyzes acetyl-CoA into acetoacetyl-CoA which is further transformed to 3-hydroxybutytyl-CoA by hbd encoded protein. These two enzymes are key enzymes in the synthesis pathway of butyl acid in Clostridium tyrobutyricum. By overexpressing thl and hbd, we enhanced the production of butyrate in Clostridium tyrobutyricum. 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]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3907
Illegal SapI.rc site found at 3731
Results
(1)Plasmid construction
The recombinant plasmid pMTL-Pthl-adhE2 (refer to: BBa_K4408008) is cleaved using XbaⅠ enzyme to obtain the linear vector. The thl fragment (1182bp) and hbd fragment (849bp) was amplified from the C. tyrobutyricum genome by PCR. The thl fragment was ligated to the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. Similar as for pMTL-Pthl-adhE2, through transformation, colony PCR and gene sequencing, we obtained the pMTL-Pthl-adhE2-thl recombinant plasmid and confirmed it. pMTL-Pthl-adhE2-thl was linearized by reverse PCR. The hbd fragment and the linear vector pMTL-Pthl-adhE2-thl were ligated by Gibson assembly method. pMTL-Pthl-adhE2-thl-hbd was transformed into E. coli JM109 strain. Colony PCR and DNA electrophoresis 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-thl-hbd by gene sequencing. The recombinant plasmid pMTL-Pthl-adhE2-thl-hbd was also verified by enzymatic cleavage using Hind III enzyme.
(2)Function of plasmid in C. tyrobutyricum
The plasmid pMTL-Pthl-adhE2-thl-hbd was transformed into C. tyrobutyricum. Protein gel electrophoresis showed that thiolysis (thl) and β-hydroxybutyryl-coenzyme (hbd) were expressed in the transformed strain (Figure 1). Fermentation experiment showed that the overexpression of these key enzymes effectively improved the growth performance of the strain, the maximum OD600 increased by 21% (Figure 2). HPLC experiment showed that the yields of butyrate and butanol were increased by 30% and 33%, respectively, in this strain (Figure 3). Since they are the precursors for butyl butyrate synthesis catalyzed by lipase, the final butyl butyrate production was up to 590 mg/L in this strain, an increase by 1.1 fold compared to the strain without thl and hbd overexpression (Figure 4).
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