Metabolic flux analysis of RQ-controlled microaerobic ethanol production by Saccharomyces cerevisiae.

Microaerobic ethanol production by Saccharomyces cerevisiae CBS 8066 was investigated at different growth rates in respiratory quotient (RQ)-controlled continuous culture. The RQ was controlled by changing the inlet gas composition by a feedback controller while keeping other parameters constant. The ethanol yield increased slightly from the anaerobic values with decreasing RQ, reaching a broad maximum at RQ 20 to 12. There was little or no glycerol production at RQ values below 17 over a wide range of dilution rates. Metabolic flux analysis revealed that a decrease in the ethanol yield at RQ 6 coincided with the cyclic, oxidative operation of the TCA cycle reactions. The model indicated that respiratory dissimilation of glucose only occurs when the oxygen uptake rate is high enough to completely substitute for glycerol formation. The cytosolic and the mitochondrial NADH balances were important for determining the flux distributions. The smallest deviations between estimated and measured product yields were obtained when the unknown co-factor requirements of a limited number of biosynthetic reactions were chosen so that the amount of excess NADH formed in biosynthesis was minimized. The biomass yield was positively correlated with the net amount of NADH reoxidized in respiration and glycerol formation, indicating that the turnover of excess NADH from biosynthesis is an important factor influencing the biomass yield under oxygen-limiting conditions. Copyright 2002 John Wiley & Sons, Ltd.