Identification of NADPH‐dependent Glutamate Dehydrogenase in Yarrowia lipolytica

The recent mapping of the genome of the nonconventional oleaginous yeast species Yarrowia lipolytica has sparked interest in its usefulness as a model for fat metabolism and as an alternative to Saccharomyces cerevisiae . However, for Yarrowia lipolytica to be more effective as a model organism, more information must be obtained about its metabolism. This study focused on the gene that encodes NADPH‐dependent glutamate dehydrogenase (GDH) in Y. lipolytica . The aims of this study were to test gene complementation in S. cerevisiae and to analyze the phenotype of gene deletion in Y. lipolytica . The NADPH‐GDH gene in Y. lipolytica ( ylGDH1/3) was identified by homology to NADPH‐dependent GDHs in S. cerevisiae ( scGDH1 and scGDH3 ). For the complementation test, the ylGDH1/3 was amplified by PCR, cloned into the S. cerevisiae pBEVY‐L vector under control of the constitutive GPD/ADH1 promoter and verified by restriction digests and sequencing. The pBEVY‐L‐ ylGDH1/3 was introduced into a S. cerevisiae null mutant strain that lacks any GDH activity. The subsequent gain of GDH activity to a level greater than that in wild type demonstrated the NADPH‐dependent GDH activity of the protein encoded by ylGDH1/3 . For gene disruption in Y. lipolytica , overlap extension PCR was used to create a ylGDH1/3 disruption cassette containing the selection marker LEU2 ( gdh1/3 :: LEU2 ) from Y. lipolytica . The cassette was transformed into Y. lipolytica forming a mutant strain via homologous recombination. PCR analysis confirmed the disruption and enzyme activity measurements indicate a significant decrease in NADPH‐dependent GDH activity in the knockout strain, which further suggests that ylGDH1/3 in Y. lipolytica encodes NADPH dependent GDH. Future investigations include examining how the NADH dependent GDH activity is affected in the mutant strain. This valuable information about the yeast's metabolism may further aid researchers in understanding the metabolic pathways, as well as using Y. lipolytica as a model to learn more about metabolic diseases such as diabetes and obesity.