3-77: Non-natural reactions to convert cellulosic biomass to fuels and chemicals

Natural conversion of cellulosic biomass to energy by free-living or gut-symbiotic microbes is slow, partly because biodegradation of dead plant tissues must be compatible with living organisms. Industrial conversion of cellulosic biomass can use a wider range of reaction conditions and intermediates to proceed at a faster rate; however, these reactions are often incomplete or produce undesirable side-products such as inhibitors. We propose the use of non-natural enzyme-catalyzed reactions that are both selective and more efficient. Specifically, we propose use of enzymes with the potential in multi-reaction conditions to release the required sugars from a complex multi-component material like cellulosic biomass. Here, we present work on an engineered perhydrolase with improved substrate recognition and that catalyzes formation of peracetic acid more efficiently than its natural counterpart by 100-fold. Peracetic acid is a strong oxidant that effectively alters lignin allowing subsequent release of sugars (saccharification) by cellulases and xylanases. We have generated up to 70 mM peracetic acid in situ, resulting in up to 45% lignin reduction and up to 97% sugar release in saccharification efficiency. At this time, we are optimizing reaction conditions to improve potential involvement of endogenous acetate groups and toward matching saccharification reaction conditions. Our goal is to allow, for the first time, a consolidated approach that includes both the pretreatment and saccharification steps.