Metabolic engineering of Clostridium beijerinckii to improve glycerol metabolism and furfural tolerance

Inefficient utilization of glycerol by Clostridium beijerinckii ( Cb ) is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration, which would in turn, alleviate the toxicity of lignocellulose-derived microbial inhibitory compounds (LDMICs, e.g., furfural), and improve the fermentation of lignocellulosic biomass hydrolysates (LBH) to butanol. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb . By overexpressing two glycerol dehydrogenase (Gldh) genes ( dhaD1 and gldA1 ) from the glycerol hyper-utilizing Clostridium pasteurianum ( Cp ) as a fused protein in Cb , we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, Cb _ dhaD1 + gldA1 achieved a 59% increase in growth, while butanol and acetone–butanol–ethanol (ABE) concentrations and productivities increased 14.0%, 17.3%, and 55.6%, respectively, relative to the control. Co-expression of dhaD1 + gldA1 and gldA1 + dihydroxyacetone kinase ( dhaK ) resulted in significant payoffs in cell growth and ABE production compared to expression of one Gldh. In the presence of 4–6 g/L furfural, increased glycerol consumption by the dhaD1 + gldA1 strain increased cell growth (> 50%), the rate of furfural detoxification (up to 68%), and ABE production (up to 40%), relative to the plasmid control. Likewise, over-expression of [( dhaD1 + gldA1 ) dhaK ] improved butanol and ABE production by 70% and 50%, respectively, in the presence of 5 and 6 g/L furfural relative to the plasmid control. Overexpression of Cp gldhs and dhaK in Cb significantly enhanced glycerol utilization, ABE production, and furfural tolerance by Cb . Future research will address the inability of recombinant Cb to metabolize glycerol as a sole substrate.