Overview
Our project aims to efficiently and massively synthesize the biofuel butyl butyrate through microbial fermentation, addressing energy-related issues on a global scale. To achieve this, we genetically engineered Clostridium tyrobutyricum (C. tyrobutyricum) to enable efficient and large-scale synthesis of butyrate and butanol. We employed a method of lipase catalyzed esterification of butyrate and butanol into butyl butyrate in which reaction the optimal butyrate-to-butanol molar ratio is 1:1. In the process of constructing C. tyrobutyricum, we used the method developed by Worldshaper-NJBIOX team last year which utilized the strain’s natural pathway for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. We further engineered C. tyrobutyricum to efficiently produce butyrate and butanol for the successive esterification into butyl butyrate through two strategies, adjusting the product molar ratio closer to 1:1 (butyrate: butanol) and co-enhancing the two products yield. The first strategy was achieved by enhancing the expression of deacetylase (Dac) in the synthesis pathway of butyrate and butanol, overexpressing CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate, and using a weaker promoter Ptkt to express adhE2. The second strategy was fulfilled by overexpressing rate-limiting enzymes (bcd and crt) in the synthesis pathway. This allowed us to develop a C. tyrobutyricum strain with a high metabolic flux for the synthesis of butyrate and butanol and a more optimal product ratio, which can be used for efficient large-scale microbial fermentation for butyl butyrate.
Our project fulfilled the following achievements.
1.Characterization of the part BBa_K3560002 (PGroES) from iGEM20_XHD-Wuhan-China
We used PRADK-PgroES as the vector and eGFP fragment amplified from the template of PCDF-1-eGFP to construct recombinant plasmid PRADK-PgroES-eGFP. This plasmid was used to express eGFP in Deinococcus radiodurans (D. radiodurans). The fluorescence intensity of the transfected strain was analyzed to characterize function of PgroES promoter in driving the expression of eGFP gene in D. radiodurans.
Our results showed that the fluorescence intensity of the transfected D. radiodurans under OD600=0.5 and OD600=1.0 was 55745和67807.3, stronger than the blank control, indicating the successful expression of eGFP.
2. mprovement of the part BBa_K4408008 (Pthl-adhE2)
Our team improved Pthl-adhE2 (BBa_K4408008) to a better version: Ptkt-adhE2.
Pthl-adhE2 is used to express adhE2 driven by Pthl promoter in C. tyrobutyricum to construct a butanol synthesis pathway based on the native butyrate synthesis pathway. Such strain had high butanol yield but reduced butyrate production with a butyrate-to-butanol molar ratio far less than 1:1. Butyrate and butanol produced by the strain are used as the precursors for the esterification of butyl butyrate, in which reaction, the molar ratio of the precursors is 1:1. So the product ratio of butyrate and butanol in the strain was not favorable for maximum butyl butyrate yield.
Our team performed promoter optimization for the expression of adhE2. We substituted Pthl promotor with a weaker promoter Ptkt 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. 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.
BBa_K4408008 (Hyperlink BBa_K4408008)
3. Plasmids constructed
We constructed pMTL-Pthl-adhE2-Dac, pMTL-Pthl-adhE2-Pcat1-cat1, pMTL-Pthl-adhE2-bcd-crt, and pMTL-Ptkt-adhE2 plasmids that can be utilized by future iGEM teams for constructing butanol synthesis pathway and enhanced butyrate synthesis pathway in Clostridium strains.
adhE2 gene encodes the alcohol/aldehyde bifunctional dehydrogenase to catalyze the conversion of butyryl-CoA to butyraldehyde and then to butanol. Dac is a gene coding a NAD-dependent deacetylase which is an enzyme removing acetyl groups from the N-terminal of protein substrates and weakening the N-terminal acetylation of proteins. Cat1 gene codes the butyryl-CoA/acetate CoA transferase. Crotonase and butyryl-CoA dehydrogenase coded by bcd and crt are considered as rate-limiting enzymes in the butyrate synthesis pathway of Clostridium.
