(29m) De Novo Biosynthesis of Butyl Butyrate Using Heterologous Lipase in Clostridium Tyrobutyricum

Butyl butyrate (BB) is widely applied to food, cosmetic, perfumes, pharmaceutical, and biofuel industries. Compared to its traditional preparation, Fischer esterification method, lipase-mediated esterification can be conducted under much more mild conditions, providing a highly specific and environment-friendly route for BB production. However, most of present studies employed supplemental exogenous lipase to synthesize the BB, which is limited by the high enzyme cost. Recently, C. tyrobutyricum has been engineered to produce butyrate and butanol, the two precursors for BB biosynthesis, at high yields and rates from glucose and xylose in fermentation. The goal of this study is to engineer C. tyrobutyricum to co-overexpress a heterologous lipase with aldehyde/alcohol dehydrogenases so the engineered mutant strain would be capable of producing BB in a one-pot process at a reduced cost. Since the source of lipase would significantly affect its reaction selectivity and rate due to the variety of active pocket, design/engineering strategies, including screening appropriate lipase for specific BB production, improving lipase expression through codon optimization and co-expressing with chaperones endoplasmic reticulum oxidoreduction 1 (ERO1), as well as enhancing its catalytic capacity by site-directed mutagenesis and lid swapping, are considered for increasing BB production. Using the HotSpot Wizard tool, we performed the substrate affinity analysis and selected Candida rugosa lipase as the catalyst. The lipase with optimized codon was cloned and expressed in C. tyrobutyricum, which produced 17.3 g/L butyl butyrate from butyrate (1.6 M) and butanol (3.3 M). Further optimization of the engineered lipase and C. tyrobutyricum for one-pot BB production from glucose/xylose in an integrated fermentation with in-situ BB separation is being investigated and will be reported in this paper.