Downloaded from www.microbiologyresearch.org by IP: 54.198.146.117 On: Sat, 25 Jun 2016 22:38:33 Microbiology (2000), 146, 239–245 Printed in Great Britain Pathways for glutamate biosynthesis in the yeast Kluyveromyces lactis Mauricio Romero, Simo  n Guzma  n-Leo  n, Cristina Aranda, Diego Gonza  lez-Halphen, Lourdes Valenzuela and Alicia Gonza  lez Author for correspondence : Alicia Gonza  lez. Tel: 52 56 22 56 31. Fax: 52 56 22 56 30. e-mail : amanjarrifisiol.unam.mx Departamento de Gene  tica Molecular, Instituto de Fisiologı  a Celular, Universidad Nacional Auto  noma de Me  xico, Apartado Postal 70-242, 04510 Mexico City, Mexico 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 KlGLT1 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 KlGDH1 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 KlGDH1 and KlGLT1 products. Keywords : glutamate biosynthesis, glutamate synthase, glutamate dehydrogenase INTRODUCTION Two pathways for ammonium assimilation and glut- amate biosynthesis have been found in a variety of micro-organisms. The first one, described by Holzer & Schneider (1957), is mediated by NADP-glutamate dehydrogenase (NADP-GDH ; EC 1414), which catalyses the reductive amination of 2-oxoglutarate to form glutamate. In an alternative pathway demonstrated by Tempest et al. (1970), glutamate is aminated to form glutamine by glutamine synthetase (GS ; EC 6312), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT ; EC 14113), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several micro-organisms (Senior, 1975 ; Hummelt & Mora, 1980 ; Bravo & Mora, 1988 ; Marque  s et al., 1992) and in higher plants (Miflin et al., 1980). Mutants affected in either NADP-GDH or GOGAT have been obtained in a number of fungal species. In the yeast Saccharomyces cerevisiae, besides the GS-GOGAT ................................................................................................................................................. Abbreviations : GDH, glutamate dehydrogenase ; GOGAT, glutamate synthase ; GS, glutamine synthetase. pathway, there are two NADP-GDH isozymes, NADP- GDH1 and NADP-GDH3, encoded by GDH1 and GDH3, respectively (Avendan  o et al., 1997). In this yeast, only the triple mutant lacking GOGAT, GDH1 and GDH3 shows a tight glutamate auxotrophy (Avendan  o et al., 1997). In Neurospora crassa and Aspergillus nidulans, the simultaneous lack of GOGAT and NADP-GDH leads to full auxotrophy, indicating that in this species, only two pathways for glutamate biosynthesis are functional (Romero & Da  vila, 1986 ; Macheda et al., 1999). Thus, S. cerevisiae is so far the only example of a micro-organism possessing three pathways for glutamate biosynthesis. In Kluyveromyces lactis, both NADP-GDH and GOGAT activities have been detected (Valenzuela et al., 1995). Mutants devoid of NADP-GDH showed a wild- type phenotype, suggesting that either the GS-GOGAT pathway plays a major role in glutamate biosynthesis or that, as found for S. cerevisiae gdh1 mutants, glutamate biosynthesis could be maintained by the combined action of the GS-GOGAT pathway and a third bio- synthetic route. To analyse this matter, we isolated a K. lactis single mutant impaired in GOGAT activity and a double mutant lacking both GOGAT and NADP-GDH. The first mutant strain did not display any growth defect phenotype whilst the double mutant was a full glutamate auxotroph. This implies that, as in N. crassa and A. 0002-3589  2000 SGM 239