Transport and Metabolism of Fumaric Acid in Saccharomyces Cerevisiae

M.V. Shah, J.J. Heijnen and W.M. van Gulik

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands   

Currently, research is focussed on the production of fumaric acid and other relevant organic acids via fermentation using Saccharomyces cerevisiae as preferred organism because of its robustness at low pH and the availability of genetic tools for metabolic engineering. Industrial fermentation of fumaric acid is advantageous to be carried out at low pH for cost effective downstream processing and reduction of waste production. However, at low cultivation pH most of the fumaric acid is present in  undissociated form, which can diffuse through the plasma membrane into the cytoplasm. In the presence of an efficient export mechanism for fumaric acid this will lead to futile cycling resulting in ATP dissipation, and will significantly reduce the product yield. Therefore it is relevant to study the importance of futile cycling in the presence of high concentration of undissociated fumaric acid and to get insight into the transport mechanism and metabolism of fumaric acid in S.cerevisiae.

In this work we studied the uptake mechanism and metabolism of fumaric acid at a pH of 3.0 in a glucose limited aerobic chemostat at a dilution rate of 0.10 h^-1 . Our study was done using the wild type strain of S. cerevisiae CEN.PK 113-7D. It was observed that fumaric acid was efficiently used as a carbon and energy source in the presence of glucose, there was  significant increase in the steady state biomass concentration and respiratory quotient with increase in biomass specific uptake rate of fumaric acid.  A linear relationship between the steady state extracellular fumaric acid concentration and the specific uptake rate of fumaric acid was observed, indicating towards the presence of passive or facilitated diffusion of the undissociated species of fumaric acid. It was found that the permeability coefficient of the cells for fumaric acid was 10 times higher than for succinic acid.  From flux analysis using stoichiometric model, we found that no additional ATP dissipation occurred at high extracellular concentrations of undissociated fumaric acid in the wild type strain. This indicates the absence of futile cycling and thus the absence of an active export mechanism of fumaric acid in wild strain. Implications of our experimental results for the industrial scale production of fumaric acid will be discussed.