The ever-increasing demand for transportation fuels, the decrease in global petroleum reserves, and the negative impact of greenhouse gases resulting from burning petroleum make renewable and sustainable biofuels an imperative for the future. First-generation biofuels produced from food crops are limited by cost and competition with food supply. Considerable effort has been made to produce fuels from lignocellulosic biomass, but the need for chemical and enzymatic pretreatment to solubilize the biomass prior to microbial bioconversion is a major economic barrier to the development of an industrial process. Here we report the metabolic engineering of a bacterium, Caldicellulosiruptor bescii, that is capable of using unprocessed switchgrass, an abundant, environmentally desirable, and economically sustainable lignocellulosic plant biomass, as feedstock to produce ethanol.

Abstract

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.