Abstract
Clostridium thermocellum is a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. Though C. thermocellum has the natural ability to convert cellulose to ethanol, the yield and titer are currently too low to be industrially relevant due to production of additional fermentation products such as organic acids (acetate, lactate, and formate), H2, and amino acids. Here, a mutant strain of C. thermocellum was constructed to remove side product formation and increase flux to ethanol. The resulting strain AG553 (C. thermocellum ∆hpt ∆hydG ∆ldh ∆pfl ∆pta-ack) was characterized on defined medium, effectively eliminating formate, acetate, and lactate production and reduce H2 production by fivefold on both model substrates and pretreated poplar and switchgrass. Ethanol yield and titer increased two- to threefold compared with the wild type on all substrates tested. Strain AG553 reached an ethanol yield of 56.1% of theoretical on 5 g/L cellobiose compared with 30.3% yield by the wild-type strain. Strain AG553 grown on 5 g/L crystalline cellulose Avicel reached an ethanol yield of 63.5% of theoretical compared with 19.9% by the wild type. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Strain AG553 was grown on cellulose loadings up to 50 g/L. While fermentation product titers did not increase in wild-type C. thermocellum beyond 5 g/L Avicel loading due to prohibitively low pH, mutant AG553 continued to show increasing ethanol titer to a loading of 20 g/L Avicel, at which point CO2 accumulation likely decreased the pH to a level too low to support growth. With the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, this strain is the best ethanol-producing CBP strain to date and will serve as a metabolic engineering platform for the production of fuels and chemicals from lignocellulosic biomass.
