Pathways for glutamate biosynthesis in the yeast Kluyveromyces lactis.
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.
Romero
Mauricio
M
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
Guzmán-León
Simón
S
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
Aranda
Cristina
C
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
González-Halphen
Diego
D
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
Valenzuela
Lourdes
L
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
González
Alicia
A
Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1.
D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005970:Glutamate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis* / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods
D018698:Glutamic Acid; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase