Metabolic pathway analysis of recombinant Saccharomyces cerevisiae with a galactose-inducible promoter based on a signal flow modeling approach

Abstract

The objective of this work was to develop a signal flow diagram-based modeling approach proposed by Endo et al. (1976) to organize the network of complex metabolic reactions occurring in the living cell, employing directed signal flow diagram in which the enzyme reaction coefficient was defined as the metabolic transfer coefficient. Using this concept, a metabolic reaction between substrate A and product B could be regarded as a signal transmittance from A to B. A very simple set of linear equations was then derived to analyze the flow directions of the carbon fluxes and the degree of activation of certain metabolic pathways within a recombinant yeast, in which the specific consumption rate of galactose, the specific rate of change of ethanol and the specific uptake rate of oxygen constituted the three input nodes of the metabolic signal transfer system. The output nodes were the specific growth rate of the cells, the specific production rate of the recombinant protein and the specific evolution rate of carbon dioxide. In this way, the effect of the culture conditions on cell growth and recombinant protein production under control of the GAL10 promoter could be characterized in terms of the metabolic pathways based on observable variables such as the cell and product concentrations, and the carbon dioxide and oxygen in the exhaust gas. This approach was successfully applied to an analysis of the metabolic pathways occurring in the fed-batch cultivation of a recombinant yeast where galactose served as both the carbon source for cell growth and as an inducer for expression of the recombinant gene. The expression period was classified into three phases—named the switch, expression, and stationary phases—on the basis of calculations using signal flow equations. In the switch phase, biomaterials for cell growth were found to be synthesized through the TCA cycle and UR loop, and less galactose entered the induction pathway. In the stationary phase, on the other hand, the formation of biomaterials for cell growth occurred mainly through the PP and EMP pathways and the TCA cycle. The degree of activation of the induction pathway was reduced in this phase. Unlike in either of the previous two phases, in the expression phase a significant amount of galactose was directed towards the induction pathway to stimulate the expression of the recombinant gene, and the PP pathway played the major role in the synthesis of biomaterials for cell growth.