Engineering cellulolytic bacterium Clostridium thermocellum to co‐ferment cellulose‐ and hemicellulose‐derived sugars simultaneously

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6‐carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo‐oligomers (with degree of polymerization of 2–7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co‐utilizing both cellulose and hemicellulose for the production of fuels and chemicals. A xylose catabolizing pathway was engineered in the naturally efficient cellulose‐degrading bacterium, Clostridium thermocellum, and enables the bacterium to co‐ferment xylose with glucose monomer, dimer (cellobiose), and polymer (Avicel, the crystalline cellulose), respectively, without carbon catabolite repression. Owing to the microbe's innate xylanolytic ability, the engineered strain can also utilize xylo‐oligomers in the presence of xylose. The engineered strain in this work unlocks the possibility toward next‐generation consolidated bioprocessing (NG‐CBP) to realize the direct and simultaneous co‐utilization of cellulose and hemicellulose without cross‐inhibition in a single strain.