Metabolic engineering of to improve glycerol metabolism and furfural tolerance.

BACKGROUND

Inefficient utilization of glycerol by () 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 .

RESULTS

By overexpressing two glycerol dehydrogenase (Gldh) genes ( and ) from the glycerol hyper-utilizing () as a fused protein in , we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, _+  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 +  and + dihydroxyacetone kinase () 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 +  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 [(+ ) ] 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 and in significantly enhanced glycerol utilization, ABE production, and furfural tolerance by . Future research will address the inability of recombinant to metabolize glycerol as a sole substrate.