Regulation of carbon metabolism in Pichia pastoris by Mxr1p and identification of novel regulators

Abstract

Pichia pastoris (a.k.a. Komagataella phaffii), a widely used yeast for recombinant protein production, is cultured under a variety of conditions ranging from shake flasks to large bioreactors employing minimal, chemically defined, and complex media containing an appropriate source of carbon such as glucose, glycerol, methanol, ethanol or acetate. P. pastoris strains GS115 (his4), a histidine auxotroph, and X33, a prototroph, are used extensively for recombinant protein production employing expression vectors containing HIS4 and ZeoR, respectively as selection markers. The latter confers resistance to the antibiotic zeocin. High growth rates are attained when metabolic intermediates in the form of yeast extract and peptone are provided in the medium. The zinc-finger transcription factor Mxr1p regulates the transcription of genes of methanol, acetate, ethanol, and amino acid metabolism in P. pastoris by binding to Mxr1p response elements (MXREs) in their promoters. While the DNA binding properties of Mxr1p are well known, the transactivation domains of Mxr1p involved in the activation of target genes of different metabolic pathways are not well characterized. In this study, we have identified a nine amino acid transactivation domain located between amino acids 365 and 373, which is essential for the transactivation of ALD6-1 encoding an aldehyde dehydrogenase during ethanol metabolism. The basic amino acid residues between 75 and 85 amino acids are essential for nuclear localization of Mxr1p in a medium containing ethanol. While the N-terminal 400 amino acids of Mxr1p are sufficient for the activation of target genes essential for ethanol metabolism, the region between 401 and 1155 amino acids is also required for the regulation of genes essential for methanol metabolism. Several novel genes whose expression is differentially regulated by Mxr1p during methanol metabolism have been identified by DNA microarray. This study demonstrates that Mxr1p is a key regulator of ethanol metabolism and provides new insights into the mechanism by which Mxr1p functions as a global regulator of multiple metabolic pathways of P. pastoris. While exploring the role of Mxr1p in acetate utilization, we serendipitously discovered that GS115 exhibits impaired growth when cultured in a complex medium containing 1% yeast extract, 2% peptone, and 2% acetate (YPA). Replenishment of histidine levels in GS115 by the expression of HIS4 encoding histidinol dehydrogenase or culturing it in YPA medium containing ≥0.5 mM histidine (YPAH) restores growth to levels equivalent to that of X33. RNA-Seq analysis indicates that the transcriptome of X33 cultured in YPA is strikingly similar to that of GS115 cultured in YPAH but significantly different from that of GS115 cultured in YPA. Thus, histidine synthesized intracellularly or provided exogenously in GS115 is likely to facilitate growth in YPA by a similar mechanism. Interestingly, nuclear genes encoding several subunits of mitochondrial ATP synthase and amino acid permeases are upregulated, while biosynthetic enzymes are downregulated in X33 and GS115 cultured in YPA and YPAH, respectively, suggesting that this increased synthesis of ATP may contribute to the growth of P. pastoris in the YPA medium. We hypothesize that high levels of acetate in YPA results in its rapid conversion to acetyl Co-A leading to depletion of ATP and growth arrest. Low energy levels may trigger the activation of a YPA-specific, histidine-mediated signaling pathway leading to altered gene expression, which facilitates energy conservation and normal growth. In this study, we have also identified the epigenetic regulator of P. pastoris and demonstrated that Gcn5, encoding a histone acetyltransferase, and the central regulator of carbon metabolism-Snf1 kinase complex is essential for the metabolism of methanol, ethanol, and acetate. Using high-throughput transcriptomics, a comparative analysis of mRNA profiles of deletion mutants of Gcn5 and Gal83 with that of transcription factors Mxr1p and Trm1p hinted at a concerted action of these regulatory proteins in regulating methanol metabolism in P. pastoris.