Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Abstract

The ability of yeast cells to grow in the presence of diverse carbon sources offers a unique opportunity to study various metabolic pathways, which is not always feasible in higher eukaryotic systems. In addition to glucose, yeast cells can utilize acetate, ethanol, glycerol, or fatty acids as the sole source of carbon, and the study of their metabolism and regulation has been one of the fascinating areas of biochemistry. The regulation of metabolic pathways of respiratory yeasts such as Pichia pastoris has not been as well studied as that of Saccharomyces cerevisiae despite the extensive use of the former for the commercial production of recombinant proteins. P. pastoris, a methylotrophic yeast, can metabolize a number of compounds, such as glycerol, methanol, acetate, and oleic acid, in addition to glucose. However, very little information is available on the transcriptional regulation of metabolic pathways other than the methanol utilization (mut) 2 pathway in this yeast species. The expression of genes of the mut pathway is regulated by at least three zinc finger proteins (1-6). Of these, methanol expression regulator 1 (Mxr1p) activates the expression of genes of the mut pathway by binding to Mxr1p response elements (MXREs) in their promoters (2, 3). Rop1p has the same DNA binding specificity as Mxr1p and functions as a repressor of genes of the mut pathway in P. pastoris cultured in nutrientrich medium containing yeast extract, peptone, and methanol (YPM) but not minimal medium containing a yeast nitrogen base, ammonium sulfate, and methanol (YNBM) (4, 5). Trm1p is also essential for the expression of genes of the mut pathway (6). However, its mechanism of action remains unknown. The differential regulation of methanol metabolism in YNBM and YPM by Mxr1p and Rop1p led us to investigate the transcriptional regulation of other metabolic pathways in cells cultured in minimal and nutrient-rich media. In this study, we demonstrate that Mxr1p regulates acetate metabolism only in cells cultured in nutrient-rich medium containing yeast extract, peptone, and acetate (YPA) but not minimal medium containing yeast nitrogen base, ammonium sulfate and acetate (YNBA). Experimental ProceduresYeast and Bacterial Strains-P. pastoris (GS115, his Ϫ ) was cultured in either nutrient-rich medium (1.0% yeast extract and 2.0% peptone) containing 2.0% glucose (YPD) or 2.0% acetate (YPA) or minimal yeast nitrogen base medium (0.17% yeast nitrogen base without amino acids and 0.5% ammonium sulfate) supplemented with 2.0% glucose (YNBD) or 2.0% sodium acetate (YNBA). Casamino acids (CAAs) and glutamate were added to YNBA medium to final concentrations of 1.0% and 0.5%, respectively. P. pastoris strains were grown at 30°C in an * This study was supported by Grant BT/PR3889/BRB/10/996/2011 from the Department of Biotechnology, New Delhi, India (to P. N. R.

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

“…The MXREs identified so far contain a core motif whose consensus sequence is 5Ј-CYCCNY-3Ј (Fig. 7A) (6,7,9,13). A sequence similar to this core MXRE is present between Ϫ151 and Ϫ146 bp (site A) of the promoter of AAT2, Ϫ213 and Ϫ208 bp of the promoter of MDH2, and Ϫ528 and Ϫ523 bp of .…”

“…1B) (3). Subcellular localization studies employing P. pastoris strain expressing a FLAG-tagged Mxr1p (13) indicates that Mxr1p localizes to the nucleus of cells cultured in YP as well as YPM (YP ϩ 2% methanol) but was cytosolic in cells cultured in YPD (YP ϩ 2% glucose) (Fig. 1C).…”

“…3A) (13,14). This trans-activation domain mediates carbon source-dependent repression and activation of Mxr1p-regulated genes (13,14).…”

“…3A) (13,14). This trans-activation domain mediates carbon source-dependent repression and activation of Mxr1p-regulated genes (13,14). To examine the role of this N-terminal trans-activation domain in the regulation of GDH2 expression, P. pastoris ⌬mxr1 strain overexpressing Mxr1p N400 was generated and named MXR1 N400 -OE (Fig.…”

“…These include the following: Mxr1p, Rop1p, Trm1p, Mit1p, and Nrg1p (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The role of these transcription factors in the regulation of amino acid metabolism has not been examined.…”