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Background  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 .    Results  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.    Conclusions  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.    Electronic supplementary material  The online version of this article (10.1186/s13068-019-1388-9) contains supplementary material, which is available to authorized users.

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