2-77: Evaluation of the Metabolic Burden of Recombinant Cellulase Expression by Saccharomyces cerevisiae in Batch Culture

In an age of dwindling fossil fuel reserves, the abundance, variety and sustainability of lignocellulosic substrates presents an interesting proposition as a feedstock for second generation biofuels.  Whereas simultaneous saccharification and fermentation could be carried out through addition of enzymes in the fermentation broth, processing costs can be substantially decreased through expression of these enzymes in a recombinant host with strong fermentative characteristics.  Previously, two recombinant strains of Saccharomyces cerevisiae Y294 were constructed expressing either an endoglucanase and β-glucosidase from Trichoderma reesi and Saccharomycopsis fibuligera (S. cerevisiae CEL 5), respectively, or these enzymes in combination with fungal cellobiohydrolases (S. cerevisiae Y118P).  Low biomass and ethanol yields during growth on phosphoric acid swollen cellulose prompted fundamental studies to quantify the metabolic burden incurred through heterologous protein expression by comparison of the kinetic and stoichiometric parameters with S. cerevisiae Y 294 harbouring an empty plasmid as control.  In bioreactor-grown batch cultures in a mineral medium with glucose as carbon source, a two-fold decrease in the maximum specific growth rate of strain CEL 5 could be correlated to a four-fold and two-fold greater expression of β-glucosidase and endoglucanase, respectively, when compared with strain Y118P.  Whereas the maximum specific rates of glucose uptake and biomass yields for these strains were similar, the greater biomass yield on glucose and glucose uptake rate of the control strain pointed towards a significant energetic demand for heterologous protein expression by the recombinant strains.