Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris

Abstract

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) ؉ glutamate (YNB ؉ Glu), or YNB ؉ aspartate (YNB ؉ Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB ؉ Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB ؉ Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB ؉ Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1. We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris. In addition to GDH2, enzymes such as aspartate aminotransferase (AAT), 2 malate dehydrogenase (MDH), and glutamine synthetase (GLN1) also play key roles in the metabolism of amino acids (4). In S. cerevisiae, AAT1 and AAT2 encode AAT localized in mitochondria (mAAT) and cytoplasm (cAAT). cAAT catalyzes the reversible conversion of glutamate and oxaloacetate into ␣-ketoglutarate and aspartate (4). The oxaloacetate thus generated is converted to malate by MDH present in the cytoplasm (cMDH) encoded by MDH2. Malate enters mitochondria via ␣-ketoglutarate-malate exchanger protein, which also transports ␣-ketoglutarate in the opposite direction. Malate is oxidized to oxaloacetate by the mitochondrial MDH (mMDH) encoded by MDH1, resulting in the formation of NADH, which enters the electron transport chain and generates energy (4). The mitochondrial oxaloacetate is converted to aspartate by mAAT, which is transported to cytoplasm via the aspartate-glutamate exchanger. Glutamate present in the cytoplasm is converted to glutamine by GLN1 (4). Saccharomyces cerevisiaeP. pastoris, a methylotrophic yeast, is extensively used for the production of recombinant proteins. Being a respiratory yeast, P. pastoris completely oxidizes sugars, avoiding formation of ethanol, and this results in efficient utilization of carbon sources yielding high biomass. During high cell density fermentation of P. pastoris, ammonium...

Effects of Deletions in <i>Pichia pastoris RTG</i> Genes on Phenotype and AOX1 Expression

“…Recent studies revealed that some amino acids (e.g. glutamate) can be used by P. pastoris as sole source of carbon and nitrogen [20]. We demonstrate that P. pastoris utilize proline in such way.…”

“…glutamate) as sole source of carbon and nitrogen [20]. To study if proline can be used by P. pastoris in such way, tr2-1-GS115 strain was placed in 10 μl of cell suspensions (10 6 , 10…”

Function and regulation of an aldehyde dehydrogenase essential for ethanol and methanol metabolism of the yeast,Komagataella phaffii

“…phaffii is cultured in minimal medium containing yeast nitrogen base (YNB) or nutrientrich medium containing yeast extract, peptone (YP) supplemented with different sources of carbon such as glucose, glycerol, methanol, ethanol, acetate, glutamate etc. Several transcription factors such as Mxr1p, Rop1p, Trm1p, Nrg1p, Mit1p, Mig1p and Mig2p (19,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) regulate the expression of target genes of specific metabolic pathways of K. phaffii. In addition to conventional sources of carbon such as glucose and glycerol, K. phaffii can utilize amino acids such as glutamate as the sole source of carbon and hence can be cultured in YNB medium containing 2% glutamate (YNB+Glu) or YP medium in which amino acids derived from peptone serve as the source of carbon (38).…”

“…In addition to conventional sources of carbon such as glucose and glycerol, K. phaffii can utilize amino acids such as glutamate as the sole source of carbon and hence can be cultured in YNB medium containing 2% glutamate (YNB+Glu) or YP medium in which amino acids derived from peptone serve as the source of carbon (38). Mxr1p regulates the expression of several enzymes required for glutamate utilization in cells cultured in YP or YNB+Glu (38). Mxr1p regulates the expression of ACS1 encoding acetyl-CoA synthetase 1 during acetate metabolism (37).…”

“…Mxr1p regulates the expression of ACS1 encoding acetyl-CoA synthetase 1 during acetate metabolism (37). The target genes of Mxr1p harbour Mxr1p response elements (MXREs) in their promoters to which Mxr1p binds and activates transcription (28)(29)(30)(31)37,38).…”