ISOLATION OF THE PICHIA-PASTORIS PYCL GENE ENCODING PYRUVATE-CARBOXYLASE AND IDENTIFICATION OF A SUPPRESSOR OF THE PYC PHENOTYPE

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon s ource and ammonium as nitrogen source. Growth was possible with aspart ate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevi siae mutants devoid of pyruvate carboxylase. In a P. pastoris strain c arrying a disrupted PpPYC1 gene we have isolated spontaneous mutants a ble to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepr essed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of th e PpPYC1 gene has been entered in the EMBL nucleotide sequence databan k: Accession Number Y11106. (C) 1998 John Wiley & Sons, Ltd.