Abstract
BackgroundInefficient 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.ResultsBy 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.ConclusionsOverexpression 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.
Highlights
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
This study was designed to achieve the following objectives: (i) to metabolically engineer Cb strains through systematic overexpression of Clostridium pasteurianum (Cp) gldh and dihydroxyacetone kinase (dhaK) to generate glycerol-utilizing Cb strains, (ii) to characterize the phenotypes of the resulting recombinant Cb strains in terms of cell growth, ABE production, and glycerol utilization, and (iii) to ascertain whether increased consumption of glycerol translates into increased rate of furfural detoxification and ABE production
Our results demonstrate that overexpression of Cp dhaD1 and gldA1 either as single or a fused protein or in combination with dhaK enhanced glycerol consumption and improved furfural tolerance by the recombinant Cb strains
Summary
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. Due to the finite nature of crude oil, coupled with environmental concerns associated with the consumption of fossil fuels, biofuels have gained significant attention over the past few decades. LB pretreatment generates LDMICs, especially furaldehydes (e.g., furfural) and phenolic compounds that limit the fermentation of lignocelluloses-derived sugars to fuels and chemicals [12]
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