Abstract
The zinc finger transcription factor Mxr1p regulates the transcription of genes involved in methanol, acetate, and amino acid metabolism of the industrial yeast Pichia pastoris (a.k.a. Komagataella phaffii) by binding to Mxr1p response elements in their promoters. Here, we demonstrate that Mxr1p is a key regulator of ethanol metabolism as well. Using transcriptomic analysis, we identified target genes of Mxr1p that mediate ethanol metabolism, including ALD6-1 encoding an aldehyde dehydrogenase. ALD6-1 is essential for ethanol metabolism, and the ALD6-1 promoter harbors three Mxr1p response elements to which Mxr1p binds in vitro and activates transcription in vivo. We show that a nine-amino acid transactivation domain located between amino acids 365 and 373 of Mxr1p is essential for the transactivation of ALD6-1 to facilitate ethanol metabolism. Mxr1N250, containing the N-terminal 250 amino acids of Mxr1p, localized to the nucleus of cells metabolizing ethanol dependent on basic amino acid residues present between amino acids 75 and 85. While the N-terminal 400 amino acids of Mxr1p are sufficient for the activation of target genes essential for ethanol metabolism, the region between amino acids 401 and 1155 was also required for the regulation of genes essential for methanol metabolism. Finally, we identified several novel genes whose expression is differentially regulated by Mxr1p during methanol metabolism 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.
Highlights
This study demonstrates for the first time that Mxr1p is required for efficient ethanol metabolism in P. pastoris and in its absence, ALD6-1-mediated conversion of acetaldehyde to acetate is impaired resulting in growth defect
Mxr1p binds to three Mxr1p response element (MXRE) in ALD6-1 promoter in vitro, and a combination of two of them is sufficient for Mxr1p-dependent expression of a reporter gene from ALD6-1 promoter in vivo during ethanol metabolism
This result is similar to that reported in an earlier study in which two MXREs in the promoter of ACS1 encoding ACS1 were shown to be required for Mxr1p-mediated transactivation during acetate metabolism [4]
Summary
To further investigate regulation of ALD6-1 by Mxr1p, we generated GS115-A and Δmxr1-A expressing Myc-tagged ALD6-1 (ALD6-1Myc) and examined ALD6-1 levels in the lysates of cells cultured in YNBE by Western blotting using anti-Myc epitope antibodies. When expressed in Δmxr1-A, it restores ALD6-1Myc levels as efficiently as Mxr1N400 (Fig. 3D) indicating that phenylalanine residues are not essential for the transactivation of ALD6-1 during ethanol metabolism.
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