Cas9 nickase-based genome engineering substantially improves cellulolytic features of Clostridium cellulolyticum

Improving enzymatic conversion of lignocellulose to fermentative sugars will boost the production of diverse chemicals and alternative biofuels from lignocellulose feedstocks.  Using the model mesophilic cellulolytic Clostridia, Clostridium cellulolyticum, we enhanced the performance of whole cell-based catalysts in cellulose degradation through genetic engineering. We applied the newly developed Cas9 nickase genome editing tool to chromosomally integrate a constitutive (P4)/cellulose-inducible promoter (Pm2112) into the major cellulosome-encoding cip-cel operon; generating an artificial six-gene operon (cel9H-cel9J-man5K-cel9M-rgl11Y-cel5N) in the genome. Two resulting strains, P4::Cel9H-5N and  Pm2112::Cel9H-5N, presented a much shorter adaption phase on cellulose and the later one consumed 15% more cellulose than the control strain. SDS-PAGE analysis of cellulose-associated proteins (mainly cellulosome components) revealed dramatic changes in band patterns especially in P4::Cel9H-5N. Enzyme assays showed that the isolated proteins of both strains had a substantial increase in endoglucanases activity and exoglucanases activity; additionally the total cellulase activity was increased in P4::Cel9H-5N. It is obvious that promoter integration significantly improved cellulolytic activities. In addition, we tried to improve cellulose utilization by overexpressing a secretary β-glucosidase (BglA) to promote cellobiose hydrolysis. The plasmid transformants presented a very high β-glucosidase activity in the supernatant and 17% higher cellulose consumption than the control. Strikingly, its overexpression in the engineered P4::Cel9H-5N and  Pm2112::Cel9H-5N further leaped cellulose consumption. These engineered strains will contribute to bioconversion of lignocellulosic biomass to value-added products.