Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc(-)) mutant was constructe d by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces c erevisiae. Its maximum specific growth rate on ethanol was identical to tha t of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose c ould be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc(-) strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h(-1) on mixtures of glucose and ethan ol. In such mixed-substrate chemostat cultures of the Pyc(-) strain, steady -slate growth could only be obtained when ethanol contributed 30% or more o f the substrate carbon in the feed. Attempts to further decrease the ethano l content of the feed invariably resulted in washout. In Pyc(-) as well as in wildtype cultures, levels of isocitrate lyase, malate synthase and phosp ho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc(-) mutant, activities of the glyoxylate cy cle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other tha n glucose repression of alternative routes for oxaloacetate synthesis preve nt growth of Pyc(-) mutants on glucose.