Difference between revisions of "Part:BBa K4408008"

Revision as of 13:45, 10 October 2023

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

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).

Figure 1 Protein gel electrophoresis of adhE2 expression in C. tyrobutyricum transformed with pMTL-Pthl-adhE2

Figure 2 Growth performance of recombinant plasmid pMTL-Pthl-adhE2 overexpression in C. tyrobutyricum

Figure 3 Butyrate and butanol yields of C. tyrobutyricum transformed with pMTL-Pthl-adhE2

Our team improved Pthl-adhE2(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.

Experiments and results

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.