| Metabolic engineering of the catabolic end-product metabolism of Thermoanaerobacterium saccharolyticum was initiated. T. saccharolyticum is an anaerobic, thermophilic bacterium that utilizes xylans and xylose, the second most prominent sugar after glucose in hardwood and herbaceous feedstocks, but not cellulose. Ethanol, acetic acid, and lactic acid are produced as fermentation products. The catabolic genes lactate dehydrogenase and phosphotransacety/aselacetate kinase were cloned and sequenced from T. saccharolyticum and subsequently used to construct suicide vectors to mediate gene knockout via homologous recombination. Lactate and acetate production was successfully eliminated via homologous recombination-mediated gene knockout as both the deletion mutant strains showed no detectable amounts of lactate and acetate at all times tested. Recombination by double cross over was confirmed via PCR and Southern blots. The lactate knockout strain exhibited a growth rate similar to the wild-type whereas the acetate knockout strain exhibited a lower growth rate and was unable to consume all the substrate presented. This may be due to saturated pathways and consequent accumulation of metabolic intermediates. The stoichiometric coefficients of products that were not knocked out—ethanol and acetate in the case of the lactate knockout and ethanol and lactate in the case of the acetate knockout—increased approximately equally when engineered strains were forced to produce two fermentation products rather than three. The results represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids and support the methodological feasibility of this goal. |
