Romero Mauricio, Guzmán-León Simón, Aranda Cristina, González-Halphen Diego, Valenzuela Lourdes, González Alicia
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
Microbiology (Reading). 2000 Jan;146 ( Pt 1):239-245. doi: 10.1099/00221287-146-1-239.
Purified glutamate synthase (GOGAT) from Kluyveromyces lactis was characterized as a high-molecular-mass polypeptide, a distinction shared with previously described GOGATs from other eukaryotic micro-organisms. Using degenerate deoxyoligonucleotides, designed from conserved regions of the alfalfa, maize and Escherichia coli GOGAT genes, a 300 bp PCR fragment from the K. lactis GOGAT gene KIGLT1 was obtained. This fragment was used to construct null 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 KIGDH1 mutant and the K. lactis null GOGAT strain were full glutamate auxotrophs. These results indicate that glutamate biosynthesis in K. lactis is afforded through the combined action of KIGDH1 and KIGLT1 products.
