(29t) Metabolic Engineering of Clostridium Tyrobutyricum for Butyl Butyrate Production from Glucose and Mannitol

Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (AdhE2), screening alcohol acyltransferases (VAATs), adjusting transcription of VAAT and adhE2, and supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25^oC with glucose in serum bottles. When the more reduced substrate mannitol was employed, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks.