Part:BBa_K4885012
Ptkt-adhE2
This is the improved part of Pthl-adhE2 (BBa_K4408008). This part is responsible for the expression of adhE2 gene with Ptkt promotor. adhE2 gene is derived from Clostridium acetobutylicum. Ptkt is a native promoter that drives the expression of transketolase (tkt) gene in C. tyrobutyricum. This part consists of Ptkt sequence (BBa_K4886005), adhE2 sequence (BBa_K1462060), and terminator sequence (BBa_K3585002). adhE2 gene encodes alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor.
Usage and biology
This is the part collection that used to engineer C. tyrobutyricum to overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt). BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.
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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]
Experiments and results
Our team improved Pthl-adhE2 (BBa_K4408008(https://parts.igem.org/Part:BBa_K4408008) to a better version.
Pthl-adhE2 is used to express adhE2 driven by Pthl promoter in Clostridium tyrobutyricum (C. tyrobutyricum) to construct a butanol synthesis pathway based on the native butyrate synthesis pathway of the strain. adhE2 encodes aldehyde dehydrogenase. Butyrate and butanol produced by the strain are used as the precursors for the esterification of butyl butyrate which is a valuable biofuel. In the esterification reaction, the molar ratio of butyrate and butanol is 1:1 which should be the optimal production ratio of butyrate and butanol in the engineered strain for maximum butyl butyrate yield. Preliminary experiments found that the introduction of adhE2 gene under the control of Pthl promoter into C. tyrobutyricum significantly reduced butyrate production, and the butanol yield was much higher than the butyrate yield. The disparity in production between the two precursors is unfavorable for the catalysis of butyl butyrate. Therefore, based on the strong promoter Pthl, our team performed promoter optimization for the expression of adhE2. In previous experiments, we identified and classified some promoters on the genome of C. tyrobutyricum. Here we selected the promoter Ptkt, which is a native promoter that drives the expression of transketolase (tkt) gene in C. tyrobutyricum. Ptkt promoter has weaker transcriptional strength than Pthl promoter. By using Ptkt, we expected to obtain an engineered strain with less production of butanol and more yield of butyrate compared with the strain expressing adhE2 under Pthl. So that our strain could achieve a better production ratio of butyrate and butanol closer to the optimal catalytic molar ratio of 1:1.
Our team substituted Pthl promotor with Ptkt promoter in the Pthl-adhE2 part (BBa_K4408008) and constructed the part Ptkt-adhE2 and the plasmid pMTL-Ptkt-adhE2. We transfected C. tyrobutyricum with pMTL-Ptkt-adhE2 and analyzed the product yields of the strain. Our experiments showed that compared with the Pthl-adhE2 part, the improved Ptkt-adhE2 part raised the butyrate-to-butanol molar ratio from 0.29 to 0.63 in C. tyrobutyricum, much closer to the optimal molar ratio of 1:1.
1.Plasmid construction and transformation: pMTL-Ptkt-adhE2
Using pMTL-Pthl-adhE2 plasmid (BBa_K4408008) as the template and Vtkt-F and Vtkt-R as the primers, a linearized vector Vtkt was amplified (7825 bp). Using the C. tyrobutyricum genome as the template and TKT-F and TKT-R as the primers, a tkt fragment was amplified (300 bp). The linearized vector Vtkt and the tkt fragment were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies (300 bp) with TKT-F and TKT-R as the primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Ptkt-adhE2 was obtained.
2. Construction of C. tyrobutyricum L319 with pMTL-Ptkt-adhE2
Ct(Ptkt-adhE2) strain was obtained by conjugation of recombinant plasmid pMTL-Ptkt-adhE2 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(Pthl-adhE2) which was a C. tyrobutyricum strain transfected with pMTL-Pthl-adhE2 was used as the control. HPLC showed that after fermentation for 215 hours (Figure 3), compared with Ct(Pthl-adhE2), Ct(Ptkt-adhE2) had increased the yield of butyrate by 66.5% and decreased the yield of butanol by 24.3%. Therefore, by using the weaker promoter Ptkt instead of Pthl, we raised the butyrate-to-butanol molar ratio from 0.29 to 0.63, much closer to the optimal ratio of 1:1. In addition, the production of acetate was reduced in Ct(Ptkt-adhE2) which was in accordance with the increased synthesis of butyrate.
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