Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

Abstract

BackgroundThe C. bescii genome does not encode an acetaldehyde/alcohol dehydrogenase or an acetaldehyde dehydrogenase and no ethanol production is detected in this strain. The recent introduction of an NADH-dependent AdhE from C. thermocellum (Fig. 1a) in an ldh mutant of this strain resulted in production of ethanol from un-pretreated switchgrass, but the thermolability of the C. thermocellum AdhE at the optimum growth temperature of C. bescii (78 °C) meant that ethanol was not produced above 65 °C.Fig. 1Proposed scheme for the pyruvate to ethanol pathway in C. thermocellum and T. pseudethanolicus 39E. a The C. thermocellum ethanol pathway. The red colored AdhE (Cthe_0423) is already expressed and tested in C. bescii [26]. b The T. pseudethanolicus 39E ethanol pathway. The green colored AdhE (Teth39_0206) and blue colored AdhB (Teth39_0218) are expressed and tested in C. bescii in this studyResultsThe adhB and adhE genes from Thermoanaerobacter pseudethanolicus 39E, an anaerobic thermophile that produces ethanol as a major fermentation product at 70 °C, were cloned and expressed in an ldh deletion mutant of C. bescii. The engineered strains produced ethanol at 75 °C, near the ethanol boiling point. The AdhB expressing strain produced ethanol (1.4 mM on Avicel, 0.4 mM on switchgrass) as well as acetate (13.0 mM on Avicel, 15.7 mM on switchgrass). The AdhE expressing strain produced more ethanol (2.3 mM on Avicel, 1.6 mM on switchgrass) and reduced levels of acetate (12.3 mM on Avicel, 15.1 mM on switchgrass). These engineered strains produce cellulosic ethanol at the highest temperature of any microorganism to date. In addition, the addition of 40 mM MOPS to the growth medium increased the maximal growth yield of C. bescii by approximately twofold.ConclusionsThe utilization of thermostable enzymes will be critical to achieving high temperature CBP in bacteria such as C. bescii. The ability to produce ethanol at 75 °C, near its boiling point, raises the possibility that process optimization could allow in situ product removal of this end product to mitigate ethanol toxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0346-4) contains supplementary material, which is available to authorized users.

Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites

“…While Caldicellulosiruptor species rely heavily on hydrogenases and fermentation to facilitate redox balance, other metabolic components also contribute to its redox metabolism. C. bescii strains expressing exogenous alcohol dehydrogenases with different redox cofactor requirements synthesized variable amounts of ethanol when grown under similar conditions (Chung et al., ). These experiments demonstrate that a better understanding of cellular redox systems is needed to more effectively engineer this biocatalyst to produce ethanol more effectively.…”

“…Some species can also produce ethanol, though this trait is not conserved across the genus. C. bescii does not natively produce ethanol as a fermentation product, but was recently engineered to produce ethanol directly from lignocellulosic biomass substrates by introducing an adhE gene, which is constitutively expressed (Chung, Cha, Guss, & Westpheling, 2014;Chung et al, 2015).…”

Genomic and physiological analyses reveal that extremely thermophilicCaldicellulosiruptor changbaiensisdeploys unique cellulose attachment mechanisms

“…Further discoveries on the capabilities of these thermostable enzymes include the unique mode of action used by the central cellulase, CelA, [8], synergistic activity in ionic liquid optimized enzyme mixtures [45,46] and the creation of designer cellulosomes from Caldicellulosiruptor catalytic domains [29]. Development of a genetics system for Caldicellulosiruptor bescii [14,16] has also expanded the scope of work with this genus, including metabolic engineering [10,12,13,50] and catalytic improvement [18,30,32,31,34,33].…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…The deletion of the maturation genes required for the nickel-iron hydrogenase showed that this enzyme was not responsible for the majority of the hydrogen production by C. bescii (14). The addition of a bifunctional alcohol dehydrogenase gene (adhE) from Clostridium thermocellum, resulting in strain JWCB032, allowed for the production of ethanol from plant biomass at 65°C (15), and production at 75°C was obtained by expressing the genes encoding AdhE and AdhB from Thermoanaerobacter pseudethanolicus 39E, although the ethanol yield was much lower (16). JWCB032 is the best ethanol-producing strain of C. bescii to date, making it thus far the most promising strain for future industrial development.…”