Target integration of an exogenous β-glucosidase enhances cellulose degradation and ethanol production in Clostridium cellulolyticum

The bacteria Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing (CBP). However, genetic engineering is necessary to improve this organism's cellulose degradation and bioconversion efficiencies to meet standard industrial requirements. In this study, CRISPR-Cas9n was used to integrate an efficient beta-glucosidase into the genome of C. cellulolyticum, disrupting lactate dehydrogenase (ldh) expression and reducing lactate production. The engineered strain showed a 7.4-fold increase in beta-glucosidase activity, a 70% decrease in ldh expression, a 12% increase in cellulose degradation, and a 32% increase in ethanol production compared to wild type. Additionally, ldh was identified as a potential site for heterologous expression. These results demonstrate that simultaneous beta-glucosidase integration and lactate dehydrogenase disruption is an effective strategy for increasing cellulose to ethanol bioconversion rates in C. cellulolyticum.