Metabolic engineering of Clostridium tyrobutyricum for production of biofuels and bio-based chemicals

The depletion of petroleum resource is aggravated by the increasing demand for fossil fuels. Moreover, the burning process of fossil fuels causes global warming and pollution. These concerns promote the intensive investigation on the development of economical processes to produce fuels and chemicals based on renewable resources. In this respect, plant biomass is the current sustainable source for the production of bio-based chemicals and biofuels.;C. tyrobutyricum is a rod-shape, gram-positive bacterium that produces butyrate, acetate, and hydrogen from various saccharides, including glucose and xylose, under strict anaerobic conditions. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. However, its role in affecting metabolic flux distribution and production of various metabolites is not well understood. In this work, a mutant with inactivated ptb gene, encoding phosphotransbutyrylase, was created by integrational mutagenesis through homologous recombination. Compared to the wild-type, the activities of phosphotransbutyrylase (PTB) and butyrate kinase (BK) in the mutant decreased 76% and 42%, respectively; meanwhile, phosphotransacetylase (PTA) and acetate kinase (AK) increased 7% and 29%, respectively. In addition, the mutant displayed a higher tolerance to butyric acid.;The fermentation conditions were optimized for the ptb mutant fermentation. The pH value was set at 6 and the initial substrate concentration was 50 g/l by using one-factor-a-time screening. The impact of ptb -disruption on the fermentation profile was studied using glucose and xylose as substrate. Compared to the wild type, the b/a ratio decreased by 34.7% for glucose and 28.1% for xylose, this could be attributed to the inactivation of ptb. The productivity of hydrogen increased 38% for glucose and 46% for xylose, which could be partially attributed to a higher specific growth rate (33% increase for glucose and 40% increase for xylose) in addition to the lower b/a ratio. The butyric acid fermentation and hydrogen production were further improved by immobilizing the cells in the fibrous-bed bioreactor to facilitate cell adaptation to attain a higher final product concentration.;The genome of C. tyrobutyricum was sequenced by using the 454 technology. Several genes encoding enzymes involved in the solvent formation pathway were annotated. Interestingly, the only missing gene for a complete butanol production route is for butyrylaldehyde dehydrogenase. Moreover, the mutants of C. tyrobutyricum showed higher tolerance to butanol than the butanol producing C. acetobutylicum. Plasmids carrying aldehyde/alcohol dehydrogenase gene aad or adhE2 from C. acetobutylicum were constructed and introduced into C. tyrobutyricum. The engineered mutants showed significant butanol production in batch fermentation with glucose as the substrate. This work demonstrated that it is feasible to use C. tyrobutyricum to produce butanol.