Total proton flux and balancing in genome-scale models: The case for the updated model of Clostridium acetobutylicum ATCC 824

Abstract

Genome-scale modeling and new strategies for constraining these models were applied in this research to find new insights into cellular metabolism and identify potential metabolic engineering strategies. A newly updated genome-scale model for Clostridium acetobutylicum, iMM864, was constructed, largely based on the previously published iRS552 model. The new model was built using a newly developed genome-scale model database, and updates were derived from new insights into clostridial metabolism. Novel methods of proton-balancing and setting flux (defined as reaction rate (mmol/g biomass/hr)) ratio constraints were applied to create simulations made with the iMM864 model approximate observed experimental results. It was determined that the following constraints must be applied to properly model C. acetobutylicum metabolism: (1) proton-balancing, (2) constraining the specific proton flux (SPF), and (3) installing proper flux ratio constraints. Simulations indicate that the metabolic shift into solventogenesis is not due to optimizing growth at different pH conditions. However, they provide evidence that C. acetobutylicum has developed strictly genetically regulated solventogenic metabolic pathways for the purpose of increasing its surrounding pH to decrease the toxic effects of high proton concentrations.

Applying a ratio constraint for the P/O ratio (a measure of aerobic respiratory efficiency) to the iAF1260 genome-scale model of E. coli K12 MG1655 was explored. Relationships were found between: (1) the P/O ratio, (2) the SPF, (3) the growth rate, and (4) the production of acetate. As was expected, higher acetate production correlates with lower P/O ratios, while higher growth correlates with higher P/O ratios. For the first time, a genome-scale model was able to quantify this relationship and targeting both the P/O ratio and the SFP is required to produce an E. coli K12 strain with either (i) maximized growth rate (and minimized acetate production) or (ii) maximized acetate production (at the expense of cell growth). A gene knockout mutant, Î ndh, was created with E. coli BL-21 to study the effects of forcibly higher P/O ratios on growth. The results suggest that a metabolic bottleneck lies with the NADH-1 complex, the NADH dehydrogenase that contributes to the generation of a proton motive force.