Pathways for glutamate biosynthesis in the yeast Kluyveromyces lactis

Purified glutamate synthase (GOGAT) from Kluyveromyces lactis was character ized as a high-molecular-mass polypeptide, a distinction shared with previo usly described COGATs from other eukaryotic micro-organisms. Using degenera te deoxyoligonucleotides, designed from conserved regions of the alfalfa, m aize and Escherichia coli GOGAT genes, a 300 bp PCR fragment from the K. la ctis GOGAT gene KIGLT1 was obtained. This fragment was used to construct nu ll GOGAT mutants of K. lactis by gene replacement. These mutants showed no growth defect phenotype and were able to grow on ammonium as sole nitrogen source. Double mutants obtained from a cross between a previously described KICDH'I mutant and the K. lactis null GOGAT strain were full glutamate aux otrophs. These results indicate that glutamate biosynthesis in K. lactis is afforded through the combined action of KICDH1 and KICLT1 products.