Metabolic engineering for enhanced furfural tolerance during cellulosic butanol fermentation by glycerol-supplemented Clostridium beijerinckii

The inability of Clostridium beijerinckii to efficiently utilize glycerol, currently experiencing a market glut due to increased biodiesel production is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration to mitigate lignocellulose-derived inhibitor ( e.g. furfural) toxicity, and improve butanol titer during fermentation of lignocellulosic biomass hydrolysates (LBH). Therefore, metabolic engineering was pursued to enhance glycerol utilization in C. beijerinckii to improve NAD(P)H regeneration and butanol production in furfural-replete LBH. Towards this goal, glycerol catabolic arsenal from the hyper-glycerol utilizing bacterium, Clostridium pasteurianum was cloned and overexpressed in C. beijerinckii. Glycerol dehydrogenase (gldh), the first enzyme in the glycerol catabolic pathway, catalyzes an NAD(P)H yielding reaction, dehydrogenation of glycerol to dihydroxyacetone (DHA) while the DHA kinase-catalyzed reaction yields a glycolytic intermediate (DHA phosphate). As a preliminary step, C. pasterianum gldh genes – dhaD1 and gldA1 were overexpressed as a fusion construct in an E. coli-Clostridium shuttle vector - pWUR460 under the control of constitutive thiolase promoter. The generated strain, C. beijerinckii-gldh was used to conduct batch acetone-butanol-ethanol (ABE) fermentation in a glucose-based medium supplemented with glycerol and 2, 3, 4, 5, or 6 g/L furfural. Fermentation profiles for all furfural concentrations show that C. beijerinckii-gldh accumulated significantly higher cell biomass (30 to 55%) when compared to the empty plasmid control. At high furfural concentrations (5 and 6 g/L), butanol production by C. beijerinckii_gldh were 10% and 46% higher, respectively, than the plasmid control. ABE concentration and productivity increased by 40.2% and 39.1% with 6 g/L furfural, and glycerol utilization increased by 44% to 70% for all furfural concentrations. Taken together, gldh overexpression in C. beijerinckii improved furfural tolerance and glycerol utilization in C. beijerinckii, thus, we infer that improved NAD(P)H regeneration stemming from glycerol catabolism supplies additional reducing power for efficient detoxification of furfural, which consequently promotes cell growth and butanol production