Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

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.Increasing demand for fuels, geopolitical instability, the limitation of global petroleum reserves, and the impact on climate change induced by greenhouse gases have increased the need for renewable and sustainable biofuels (1–5). First-generation biofuels produced from food crops, such as corn, are limited by cost and competition with food supply (6, 7). Switchgrass is a perennial grass native to North America, and its high productivity on marginal farmlands and low agricultural input requirements make it an attractive feedstock for the production of biofuels and biochemicals (8). A yield of 36.7 Mg⋅ha⁻¹ was achieved in field trials in Oklahoma (9), and switchgrass has the potential to produce 500% or more energy than is used for its cultivation (10). The use of abundant lignocellulosic plant biomass as feedstock is environmentally desirable and economically essential for enabling a viable biofuels industry (11). Current strategies for bioethanol production from lignocellulosic feedstocks require three major operational steps: physicochemical pretreatment, enzymatic saccharification, and fermentation (Fig. 1) (6, 12). Pretreatment and enzymatic hydrolysis represent substantial cost and it is estimated that the use of cellulolytic microbes for consolidated bioprocessing and eliminating pretreatment would reduce bioprocessing costs by 40% (2). Considerable effort has been made to develop single microbes capable of both saccharification and fermentation to avoid the substantial expense of using saccharolytic enzyme mixtures (13). Heterologous expression of saccharolytic enzymes has been demonstrated in a number of organisms, including Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli, and Bacillus subtilis to ferment various model cellulosic and hemicellulosic substrates (13–15). Although these approaches have resulted in progress in cellulose utilization, the overall enzyme activity is still very low compared with that of naturally cellulolytic organisms and the rates of hydrolysis are not sufficient for an industrial process (13).Open in a separate windowFig. 1.Comparison of bioethanol production strategies and a predicted fermentative pathway in C. bescii. Depiction of “single step bioprocessing” accomplished by engineered C. bescii. CBP, consolidated bioprocessing.High-temperature fermentations facilitate biomass deconstruction and may reduce contamination and volatilize toxic products, such as alcohols. Clostridium thermocellum and Thermoanaerobacterium saccharolyticum have been used in mixed culture fermentations successfully for laboratory-scale demonstration of first-generation consolidated bioprocessing (13, 16) (Fig. 1). C. thermocellum is one promising candidate for consolidated bioprocessing because it is naturally cellulolytic, able to hydrolyze cellulose at 2.5 g⋅L⁻¹⋅h⁻¹, and produces ethanol as one fermentation product, but it has not yet been engineered to produce ethanol at high yield and lacks the ability to ferment hemicellulosic sugars (13, 17). Caldicellulosiruptor bescii, on the other hand, is the most thermophilic cellulolytic bacterium so far described, growing optimally at ∼80 °C with the ability to use a wide range of substrates, such as cellulose, hemicellulose, and lignocellulosic plant biomass without harsh and expensive chemical pretreatment (17, 18), efficiently fermenting both C₅ and C₆ sugars derived from plant biomass (17, 18). C. bescii uses the Embden–Meyerhof–Parnas pathway for conversion of glucose to pyruvate, and the predominant end-products are acetate, lactate, and hydrogen (Fig. 2) (18). A mutant strain of C. bescii (JWCB018) was recently isolated in which the lactate dehydrogenase gene (ldh) was disrupted spontaneously via insertion of a native transposon (19, 20). A complete deletion of ldh was also engineered (21), and this strain no longer produced lactate, instead diverting metabolic flux to additional acetate and H₂, demonstrating the utility of the newly developed tools to provide a platform for further strain engineering. The recent development of genetic methods for the manipulation of this organism (19, 21, 22) opens the door for metabolic engineering for the direct conversion of unpretreated plant biomass to liquid fuels, such as ethanol, via “single step bioprocessing” (Fig. 1).Open in a separate windowFig. 2.Overview of C. bescii fermentative pathways for bioconversion of hexose sugars. Pathway 1 (blue) results in 2 mol of acetic acid and 4 mol of H₂ per mole of glucose. Pathway 2 (green) produces 2 mol of lactic acid per mole of glucose. Pathway 3 (red) is a new pathway resulting from heterologous expression of the C. thermocellum adhE gene to synthesize 2 mol of ethanol per mole of glucose.