2.3. A systemic approach to identify key reactions for ethanol production
In this research, a systemic approach was applied for S. cerevisiae to find candidate reactions for up and down-regulation for the overproduction of ethanol. As S. cerevisiae is the main industrial ethanologenic microorganism and the production rate of ethanol is dependent on pH, we assumed that by recognizing the key reactions for ethanol production at optimal pH, not only can we maintain the cell at optimum pH, but also increasing ethanol production indirectly.
Initially, the optimal pH level for ethanol production was identified based on a double robustness analysis determining the sensitivity of growth to proton exchange and ethanol production fluxes. Then, maximum ethanol production and flux distribution at optimal growth in the selected range of proton exchange rate for the desired pH level were determined. The absolute Pearson correlation coefficient of 0.95 (p-value of 0.05) between the flux of each reaction and ethanol production rate was used to determine key reactions coupled with ethanol production for optimal growth at the desired pH. If the coefficient was positive (negative), the reaction was selected for up (down) regulation. The Pearson correlation coefficient (between the flux of each key reaction and growth rate) was also applied to determine growth associated reactions. For evaluation of the role and importance of each key reaction on metabolic differentiation, fluxes of the key reactions at pH levels of 5, 6 and 7 were applied to cluster by PCA. Figure 2 indicates the flowchart of the systemic approach for finding the key reactions for ethanol overproduction and identification of the effect of pH on the metabolism.