P169: Genetic Manipulation of the pyruvate metabolic pathway in Caldicellulosiruptor bescii: deletion of the L-lactate dehydrogenase gene results in a significant increase in acetate and hydrogen production from biomass

Members of the anaerobic hyperthermophilic 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 and acetate as fermentation products, and while some Caldicellulosiruptor strains produce small amounts of hydrogen, C. bescii does not making it an attractive background to examine the effects of metabolic engineering. Genetic methods for manipulation of members of this genus have recently been developed in our laboratory and we report the first targeted deletion - the gene encoding lactate dehydrogenase (ldh). A deletion was constructed on a non-replicating plasmid and introduced into the C. bescii chromosome by marker replacement. The deletion failed to produce detectable levels of lactate confirming predictions made about the role of this predicted lactate dehydrogenase gene pyruvate metabolism and grew better than the wild type on both carbohydrate substrates, maltose and cellobiose, and non-pretreated switchgrass. A model derived from genomic analysis predicted that loss of lactate biosynthesis which consumes NADH, would result in excess NADH and we show that the deletion strain apparently channels excess NADH to the production of hydrogen. The fact that this strain grew better than the wild type on non-pretreated biomass and that the predictions made from genomic analysis were true on both carbohydrates and switchgrass suggests that these organisms will provide a new paradigm for consolidated bioprocessing: the use of Caldicellulosirutor for direct conversion of biofuels and bioproducts from biomass.