Production of fungal α-amylase by Saccharomyces kluyveri in glucose-limited cultivations
Abstract Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. α-Amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 μ plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active α-amylase in both cases. The S. cerevisiae 2 μ plasmid was found to be stable in S. kluyveri as evaluated by a constant α-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted α-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h −1 (4.8–5.7 mg (g dry weight) −1 ). At a dilution rate of 0.3 h −1 the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l −1 and the α-amylase concentration reached 320 mg l −1 . Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.