Abstract
BackgroundMembers of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment. C. bescii produces primarily lactate, acetate and hydrogen as fermentation products, and while some Caldicellulosiruptor strains produce small amounts of ethanol C. bescii does not, making it an attractive background to examine the effects of metabolic engineering. The recent development of methods for genetic manipulation has set the stage for rational engineering of this genus for improved biofuel production. Here, we report the first targeted gene deletion, the gene encoding lactate dehydrogenase (ldh), for metabolic engineering of a member of this genus.ResultsA deletion of the C. bescii L-lactate dehydrogenase gene (ldh) was constructed on a non-replicating plasmid and introduced into the C. bescii chromosome by marker replacement. The resulting strain failed to produce detectable levels of lactate from cellobiose and maltose, instead increasing production of acetate and H2 by 21-34% relative to the wild type and ΔpyrFA parent strains. The same phenotype was observed on a real-world substrate – switchgrass (Panicum virgatum). Furthermore, the ldh deletion strain grew to a higher maximum optical density than the wild type on maltose and cellobiose, consistent with the prediction that the mutant would gain additional ATP with increased acetate production.ConclusionsDeletion of ldh in C. bescii is the first use of recently developed genetic methods for metabolic engineering of these bacteria. This deletion resulted in a redirection of electron flow from production of lactate to acetate and hydrogen. New capabilities in metabolic engineering combined with intrinsic utilization of lignocellulosic materials position these organisms to provide a new paradigm for consolidated bioprocessing of fuels and other products from biomass.
Highlights
Members of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment
Deletion of lactate dehydrogenase from the C. bescii chromosome We recently reported a method for DNA transformation and marker replacement in Caldicellulosiruptor bescii based on uracil prototrophic selection [14,27,28]
To confirm the ldh deletion in JWCB017, the region of the ldh locus was amplified by PCR using primers outside of the plasmid regions of homology used to construct the deletion (Figure 2B)
Summary
Members of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment. Members of the genus Caldicellulosiruptor are able to ferment all primary C5 and C6 sugars from plant biomass and are the most thermophilic cellulolytic bacteria known, with growth temperature optima between 78°C ~ 80°C [10]. They can grow on and degrade biomass containing high lignin content as well as highly crystalline cellulose without conventional pretreatment [11,12,13], raising the possibility of further economic improvement of biofuel production from plant biomass by reducing or eliminating the pretreatment step. Recent advances have enabled genetic transformation of Caldicellulosiruptor bescii [14], opening the possibility of metabolic engineering for improved biofuel production in this genus
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