Abstract
Background: 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. 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.
Results: A 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.
Conclusions: Deletion 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.
