The ability to provide for the fuel and energy needs of the world's growing population has emerged as one of the great challenges of this century. Current fuel and energy needs are primarily met by non-renewable fossil fuels, a source that is both unsustainable and increasingly cost-inefficient. Therefore, new approaches to solving the world's energy needs are required to address these mounting concerns.
Among forms of plant biomass, lignocellulosic biomass is particularly well-suited for energy applications because of its large-scale availability, low cost, and environmentally benign production. In particular, many energy production and utilization cycles based on cellulosic biomass have very low greenhouse gas emissions on a life-cycle basis. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of biomass feedstocks to conversion into useful products. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol or other end-products including lactic acid and acetic acid. In order to convert the carbohydrate fractions, the cellulose or hemicellulose must ultimately be converted, or hydrolyzed, into monosaccharides. The hydrolysis of cellulose and hemicellulose has historically proven to be problematic.
Cellulose digesting anaerobic bacteria are of great potential utility because they can be used to produce ethanol or other fuels from abundant substrates such as forestry, municipal and agricultural waste. However, it has been challenging to realize the potential utility of biomass because of difficulty in the genetic manipulation of anaerobic bacteria and a lack of understanding of their metabolic biochemistry. Genome sequence data and recent advances in biotechnological tools for genetic modification of Clostridium thermocellum and other similar organisms have made it possible to make progress in the utilization of biomass for fuel, but the great complexity of metabolism makes it difficult to achieve a desired outcome such as near theoretical ethanol yield from cellulosic substrates.
Many microorganisms can metabolize glucose, cellulose or cellodextrins anaerobically, but they vary in the pathways utilized and the products generated. It has been demonstrated in genetically modified Thermoanaerobacterium saccharolyticum that glucose and cellobiose can be fermented to ethanol at very close to theoretical yield, but similar genetic manipulations in Clostridium thermocellum have not had the same outcome. Argyros et al. “High ethanol titers from cellulose using metabolically engineered thermophilic, anaerobic microbes.” Appl. Env. Microbiol., 2011 doi:10.1128/AEM.00646-11 (epub ahead of publication).
Clostridium thermocellum has both cellulolytic and ethanologenic fermentation capabilities and can directly convert a cellulose-based substrate into ethanol. However, C. thermocellum possesses a branched carbon utilization pathway that generates undesirable products, and thus its yield of ethanol is low. Furthermore, C. thermocellum is not as amenable to manipulation for ethanol production as T. saccharolyticum. The difficulty in manipulating C. thermocellum for ethanol production is exemplified more clearly when the carbon utilization pathways from C. thermocellum and T. saccharolyticum are compared. In homoethanologenic T. saccharolyticum, the carbon atoms from glucose flow down a linear central metabolic pathway to ethanol (FIG. 1A). In C. thermocellum, a different set of enzymes is present and thus the carbon utilization pathway (FIG. 1B) is different that the carbon utilization pathway in T. saccharolyticum. The difference in the carbon-utilization pathways of C. thermocellum compared to T. saccharolyticum makes it infeasible to produce ethanol at theoretical yield with the same modifications.
Many enzymes in carbon-utilizing metabolic processes use a nicotinamide adenine dinucleotide as a cofactor. There are two common types of nicotinamide adenine dinucleotide cofactors, NAD+ and NADP+. Each can exist in a reduced or oxidized form. In order to maintain steady state, each cofactor involved in a reaction must be regenerated at the same rate it is consumed. In other words, the cell must be reduction-oxidation (“redox”) balanced. Enzymes are typically specific for (i.e. react with) either the phosphorylated (NADP+, NADPH) or non-phosphorylated (NAD+, NADH) nicotinamide cofactors. The specificity of an enzyme can sometimes be switched from one nicotinamide cofactor to the other by mutations in the cofactor binding region of the protein. It is also possible to find different isoforms of an enzyme that carry out the same enzymatic activity, but use different cofactors (e.g. NAD+ instead of NADP+). Isoforms with altered cofactor specificity may be found for example in different species.
The T. saccharolyticum oxidation-reduction reactions in the metabolic pathway from cellobiose to ethanol are:
(1) D-glyceraldehyde 3-phosphate+phosphate+NAD+=3-phospho-D-glyceroyl phosphate+NADH+H+ (catalyzed by glyceraldehyde-3-phosphate dehydrogenase)
(2) pyruvate+CoA+oxidized ferredoxin=acetyl-CoA+CO2+reduced ferredoxin+H+ (catalyzed by pyruvate oxidoreductase)
(3) reduced ferredoxin+NADH+2 NADP++H+=oxidized ferredoxin+NAD++2 NADPH (catalyzed by NADH-dependent reduced ferredoxin:NADP+oxidoreductase)
(4) acetyl-CoA+NADPH+H+=acetaldehyde+CoA+NADP+ (catalyzed by acetaldehyde dehydrogenase)
(5) acetaldehyde+NADPH+H+=ethanol+NADP+ (catalyzed by alcohol dehydrogenase)
Reactions 1-5 above are redox and cofactor balanced. A single polypeptide called AdhE contains both catalytic activities of steps 4 and 5. Activity of AdhE is detectable with both NADH and NADPH cofactors (See Shaw et al., “Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield.” PNAS 2008. 105(37): 13769-74). In C. thermocellum, activity can be detected for both cofactors in the alcohol dehydrogenase reaction, but the aldehyde dehydrogenase reaction is specific to NADH only (See Brown et al., “Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum.” PNAS 2011. 108(33): 13753-7 and Rydzak et al., “Growth phase-dependent enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405.” J. of Biotech. 2009. 104(3-4): 169-75). Therefore, reaction 4 above cannot occur in C. thermocellum. Reaction 4 can occur with NADH as the cofactor, but use of NADH would lead to an overabundance of NADPH and depletion of NADH in the cell. The oxidation-reduction reactions in C. thermocellum in the pathway from cellobiose to ethanol are the same as 1-5 above, but with the addition of two more:
(6) oxaloacetate+NADH+H+=malate+NAD+ (catalyzed by malate dehydrogenase)
(7) malate+NADP+=pyruvate+CO2+NADPH (catalyzed by malic enzyme)
The net effect of these two additional reactions in C. thermocellum is that electrons are transferred from NADH to NADPH. This leads to a further accumulation of NADPH and makes the pathway from cellobiose to ethanol unbalanced for cofactors and therefore infeasible in this configuration. As a result, C. thermocellum strains lacking the ability to make other end products (e.g. mutants for lactate dehydrogenase and phosphotransacetylase) show poor ethanol productivity and secrete amino acids that consume NADPH during their biosynthesis.
Consequently, in order to optimize ethanol production in C. thermocellum, there is a need for mutant strains of C. thermocellum that are reduction-oxidation and cofactor balanced.
The present invention relates to cellulose-digesting organisms that have been genetically modified to allow the production of ethanol at high yield by changing cofactor usage and/or production at key steps of central metabolism.