Post-transcriptional regulation of glutamate metabolism of Pichia pastoris and development of a glutamate-inducible yeast expression system
Pichia pastoris harbours a unique glutamate utilization pathway in which glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the conversion of glutamate to -ketoglutarate, oxaloacetate, and phosphoenolpyruvate respectively in the cytosol. GDH2 and PEPCK are glutamate-inducible enzymes and their synthesis is regulated post-transcriptionally by Rtg1p, a cytosolic basic helix-loop-helix protein via Rtg1p response elements located downstream of TATA box of GDH2 and PEPCK promoters. Glutamate-inducible synthesis of PEPCK is abrogated in{Delta} gdh2 and{Delta} aat2. -ketoglutarate induces PEPCK synthesis in{Delta} gdh2 but not{Delta} aat2. We propose that oxaloacetate derived from glutamate is the inducer of PEPCK synthesis. Enzymes of glutamate utilization pathway are synthesized during carbon starvation and they enable P. pastoris to overcome nutritional stress. Finally, green fluorescent protein can be synthesized efficiently from GDH2 and PEPCK promoters using food-grade monosodium glutamate as inducer indicating that the post-transcriptional regulatory circuit described here can be exploited for the development of glutamate-inducible P. pastoris expression system.