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Abstract/Overview

Our project aims to efficiently synthesize the biofuel butyl butyrate through microbial fermentation. To achieve this, we genetically engineered Clostridium tyrobutyricum (C. tyrobutyricum) to enable efficient and large-scale synthesis of butyrate and butanol. We utilized its natural pathway for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. We employed a method of lipase catalyzed esterification of butyrate and butanol into butyl butyrate.
Our project was based on the project of Worldshaper-NJBIOX team last year. They utilized the natural pathway of C. tyrobutyricum for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. Based on this strain, we further engineered C. tyrobutyricum to efficiently produce butyrate and butanol with a better product molar ratio (closer to 1:1) for the successive esterification of these two precursors into butyl butyrate. To achieve this goal, we successfully constructed 8 parts to engineer C. tyrobutyricum. We overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885000 and BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885001, BBa_K4885005 and BBa_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt). BBa_K4885003-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.

Table 1 Part list