Metabolic engineering for enhanced furfural tolerance during cellulosic butanol fermentation by glycerol-supplemented Clostridium beijerinckii
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Date
2016-06-17
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Abstract
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.
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C. Agu, V. C. Ujor, T. C. Ezeji. Metabolic Engineering to Enhance Furfural Tolerance during Cellulosic Butanol
Fermentation by Glycerol-Supplemented Clostridium beijerinckii. Poster Presentation. ASM Microbe, Boston, June 16-20, 2016.