The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (T b = 56°C), ideally produced from lignocellulosic biomass. Caldicellulosiruptor bescii (T opt 78°C), an extremely thermophilic fermentative bacterium naturally capable of deconstructing and fermenting lignocellulose, was metabolically engineered to produce acetone. When the acetone pathway construct was integrated into a parent strain containing the bifunctional alcohol dehydrogenase from Clostridium thermocellum, acetone was produced at 9.1 mM (0.53 g/L), in addition to minimal ethanol 3.3 mM (0.15 g/L), along with net acetate consumption. This demonstrates that C. bescii can be engineered with balanced pathways in which renewable carbohydrate sources are converted to useful metabolites, primarily acetone and H 2 , without net production of its native fermentation products, acetate and lactate.
Background: Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. Results: Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R 2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. Conclusions: Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.
The extremely thermophilic cellulolytic bacterium,
Caldicellulosiruptor bescii
, degrades plant biomass at high temperatures without any pretreatments and can serve as a strategic platform for industrial applications. The metabolic engineering of
C. bescii
, however, faces potential bottlenecks in bio-based chemical productions.
To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical.
Caldicellulosiruptor bescii
and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by
C. bescii
to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium.
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