Journal of Biotechnology 54 (1997) 161 – 174 Metabolic flux distributions in recombinant Saccharomyces cereisiae during foreign protein production Sha Jin, Kaiming Ye, Kazuyuki Shimizu * Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820, Japan Received 13 February 1997; accepted 14 February 1997 Abstract A stoichiometric flux balancing analysis was applied to the recombinant yeast cultivation to examine the cellular physiology and relationship between the production of heterologous protein and metabolic fluxes. The fluxes in the metabolic pathway within a recombinant S. cereisiae grown on galactose alone or mixtures of galactose and ethanol medium were calculated. It is found that an amplification of the PP (Pentose Phosphate) pathway activity resulted in an improvement of the foreign protein expression and cell yield on ATP. The carbon source used for biosynthesis from TCA cycle in the exponential growth phase was 2 and 5-fold higher, respectively, as compared with that in the late exponential growth phase and stationary phase in batch culture with galactose minimum medium. The metabolism of ethanol together with galactose in the recombinant cell looks like increasing the flux from Acetyl-CoA to TCA cycle, and amplifying the flux directing the synthesis of various kinds of precursors such as amino acids and nucleic acid which are necessary for production of a foreign protein. Metabolic flux distribution analysis also shows that the ATP synthesis rate under substrate-level phosphorylation in the mixed carbon source cultivation was lower than that in the sole carbon source (galactose) during the expression of foreign protein. However, the total ATP production rate was higher in the mixed carbon source cultivation. © 1997 Elsevier Science B.V. Keywords: Flux analysis; Metabolic engineering; Recombinant yeast culture; Promoter GAL10; Stoichiometric flux balancing analysis 1. Introduction There has been increasing interest in using bio- logical means to produce biochemicals since the success of gene cloning about two decades ago. Increase or redirection of metabolic flux or alter- ation of metabolic pathway can be realized by using recombinant DNA technology. Increasing the titer of the rate-controlling enzymes by gene cloning is believed to be an effective way to increase the flux through the metabolic pathway (Delgado and Liao, 1991). Diaz-Ricci et al. (1992) * Corresponding author. 0168-1656/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.