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

Umakant Sahu,Pundi N Rangarajan

doi:10.1074/jbc.m116.740191

Umakant Sahu, Pundi N Rangarajan

Open Access

https://doi.org/10.1074/jbc.m116.740191

Copy DOI

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.

Highlights

By GDH2 acts as a catabolic enzyme by converting glutamate to ␣-ketoglutarate and NH4ϩ in the presence of NADH
We examined the ability of P. pastoris GS115 strain to grow in media such as yeast nitrogen base (YNB) without amino acids and 0.5% ammonium sulfate supplemented with 2.0% glucose (YNBD), 1.0% glutamate (YNB ϩ Glu), 1% aspartate (YNB ϩ Asp) or YNB ϩ Glu without ammonium sulfate
Subcellular localization studies employing P. pastoris strain expressing a FLAG-tagged Mxr1p [13] indicates that Mxr1p localizes to the nucleus of cells cultured in yeast extract and peptone (YP) as well as YPM (YP ϩ 2% methanol) but was cytosolic in cells cultured in YP medium containing 2.0% glucose (YPD) (YP ϩ 2% glucose) (Fig. 1C)

Summary

Results

Enzymes Essential for the Utilization of Amino Acids as the Sole Source of Carbon by P. pastoris and Regulation of Their Biosynthesis by Mxr1p—The ability of P. pastoris to utilize amino acids as the sole source of carbon and nitrogen has not been investigated. The fact that expression of GDH2 results in the restoration of growth of ⌬gdh (Fig. 2F) but not ⌬mxr strain indicates that Mxr1p may regulate the expression of other enzymes that are required for utilization of amino acids as the sole source of carbon in P. pastoris. To examine whether mAAT and cAAT are required for the utilization of amino acids as the sole source of carbon in P. pastoris, they were expressed as Myc-tagged proteins, and their localization in mitochondria and cytoplasm, respectively, was confirmed by immunofluorescence using anti-Myc tag antibodies (Fig. 5A). Overexpression of Mxr1p in ⌬mxr strain results in the restoration of synthesis of several key enzymes involved in amino acid metabolism in cells cultured in YP as well as YNB ϩ Glu media. There is good correlation between promoter occupation in vivo and transcriptional activation by Mxr1pN400 (Fig. 7J)

Discussion

Plasmids used in this study

YP medium