Engineering redox-balanced ethanol production in the cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii

Journal Article · Sat Dec 01 00:00:00 EST 2018 · Metabolic Engineering Communications

DOI:https://doi.org/10.1016/j.mec.2018.e00073· OSTI ID:1439267

Williams-Rhaesa, Amanda M.; Rubinstein, Gabriel M.; Scott, Israel M.; Lipscomb, Gina L.; Poole, II, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

Caldicellulosiruptor bescii is an extremely thermophilic cellulolytic bacterium with great potential for consolidated bioprocessing of renewable plant biomass. Since it does not natively produce ethanol, metabolic engineering is required to create strains with this capability. Previous efforts involved the heterologous expression of the gene encoding a bifunctional alcohol dehydrogenase, AdhE, which uses NADH as the electron donor to reduce acetyl-CoA to ethanol. Acetyl-CoA produced from sugar oxidation also generates reduced ferredoxin but there is no known pathway for the transfer of electrons from reduced ferredoxin to NAD in C. bescii. Herein, we engineered a strain of C. bescii using a more stable genetic background than previously reported and heterologously-expressed adhE from Clostridium thermocellum (which grows optimally (Topt) at 60 °C) with and without co-expression of the membrane-bound Rnf complex from Thermoanaerobacter sp. X514 (Topt 60 °C). Rnf is an energy-conserving, reduced ferredoxin NAD oxidoreductase encoded by six genes (rnfCDGEAB). It was produced in a catalytically active form in C. bescii that utilized the largest DNA construct to be expressed in this organism. The new genetic lineage containing AdhE resulted in increased ethanol production compared to previous reports. Ethanol production was further enhanced by the presence of Rnf, which also resulted in decreased production of pyruvate, acetoin and an uncharacterized compound as unwanted side-products. Using crystalline cellulose as the growth substrate for the Rnf-containing strain, 75 mM (3.5 g/L) ethanol was produced at 60 °C, which is 5-fold higher than that reported previously. This underlines the importance of redox balancing and paves the way for achieving even higher ethanol titers in C. bescii.

• Research Organization: Oak Ridge National Lab (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 1439267
• Alternate ID(s): OSTI ID: 1629711
• Journal Information: Metabolic Engineering Communications, Journal Name: Metabolic Engineering Communications Vol. 7 Journal Issue: C; ISSN 2214-0301
• Publisher: Elsevier
• Country of Publication: Netherlands
• Language: English