Development of metabolic flux analysis and antisense RNA technologies as tools for the metabolic engineering of Clostridium acetobutylicum ATCC 824

A stoichiometric model describing the metabolism of solventogenic clostridia has been improved. The singularity in the stoichiometric matrix that prevented the calculation of in vivo fluxes was resolved by developing a constraint using information from in vitro studies of the CoA transferase to relate the acetate and butyrate uptake fluxes. Boundary constraints were also incorporated to enforce thermodynamic constraints. Subsequently, a software package was developed utilizing a model independent heuristic global optimization approach to solve the resultant nonlinear problem. The validity of the nonlinear constraint was established by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles.; Next, the improved model was used to analyze fermentation data from several strains of C. acetobutylicum ATCC 824. Metabolic flux analysis revealed previously obscured roles for the acid formation pathways. The acetate formation pathway supported significant fluxes throughout the stationary phase while the butyrate formation pathway was found to uptake butyrate. Metabolic flux analysis of a butyrate kinase knockout strain revealed that the enhanced solvent production characteristic was due to early induction of butanol formation as opposed to substantially higher butanol formation fluxes. Analysis also revealed enhanced uptake of acetate and substantially reduced butyrate formation and uptake.; Antisense RNA strategies for the metabolic engineering of C. acetobutylicum were assessed by targeting butyrate formation enzymes for down-regulation. Strain 824(pRD4) was developed to down-regulate butyrate kinase activity and exhibited 85%--90% lower BK and acetate kinase (AK) activities. It also exhibited 45%--50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase (PTA) specific activities due to homology. Strain 824(pRD4) exhibited earlier induction of solventogenesis resulting in 50% and 35% higher final titers of acetone and butanol, respectively. Strain 824(pRD1) was developed to down-regulate PTB enzyme levels. It exhibited 70% and 80% lower PTB and BK activities, respectively, and 300% higher levels of a lactate dehydrogenase. Growth yields of 824(pRD1) were also decreased by 28%. While levels of acids were unaffected in 824(pRD1) fermentations, acetone and butanol titers were reduced by 96% and 75%, respectively. This reduction in solvent production by 824(pRD1) was compensated by ∼ 100 fold higher levels of lactate levels of lactate production.