Characterization of the transactivation and nuclear localization functions of Pichia pastoris zinc finger transcription factor Mxr1p
2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2021.101247
ISSN1083-351X
AutoresAditi Gupta, Kamisetty Krishna Rao, Umakant Sahu, Pundi N. Rangarajan,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoThe 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. 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. Pichia pastoris (a.k.a. Komagataella phaffii) is a methylotrophic yeast capable of utilizing methanol as a sole carbon source. The methanol utilization pathway enzyme alcohol oxidase, encoded by AOX1, constitutes 30% of the total soluble protein. This strong induction under methanol conditions has led to the development of a methanol-inducible AOX1-based promoter for commercial production of heterologous proteins (1Cereghino J.L. Cregg J.M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol. Rev. 2000; 24: 45-66Crossref PubMed Google Scholar, 2van der Klei I.J. Yurimoto H. Sakai Y. Veenhuis M. The significance of peroxisomes in methanol metabolism in methylotrophic yeast.Biochim. Biophys. Acta. 2006; 1763: 1453-1462Crossref PubMed Scopus (152) Google Scholar). It is a respiratory yeast, suggesting that in high glucose conditions biomass production through tricarboxylic acid cycle is favored over the conventional ethanol generation route followed in other yeasts like Saccharomyces cerevisiae because of the increased glycolytic flux. Consequently, the high biomass generation in the bioreactors dramatically increases the product yields, making them industrially more relevant (3Peña D.A. Gasser B. Zanghellini J. Steiger M.G. Mattanovich D. Metabolic engineering of Pichia pastoris.Metab. Eng. 2018; 50: 2-15Crossref PubMed Scopus (100) Google Scholar). Another remarkable feature of this yeast is the ability to utilize a plethora of carbon compounds as the sole source of carbon. These include glucose, glycerol, ethanol, acetic acid, oleic acid, sorbitol, and amino acids such as glutamate. The genes encoding key enzymes of these metabolic pathways are primarily regulated at the transcriptional level (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Sahu U. Rangarajan P.N. 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.J. Biol. Chem. 2016; 291: 20588-20601Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In the case of methanol metabolism, several transcriptional regulators, such as Mxr1p, Trm1p, Mit1p, Rop1p, Mig1p, Mig2p, and Nrg1p, regulate the transcription of AOX1 gene encoding alcohol oxidase 1 (6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar, 7Sahu U. Krishna Rao K. Rangarajan P.N. Trm1p, a Zn(II)2Cys6-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.Biochem. Biophys. Res. Commun. 2014; 451: 158-164Crossref PubMed Scopus (34) Google Scholar, 8Wang X. Wang Q. Wang J. Bai P. Shi L. Shen W. Zhou M. Zhou X. Zhang Y. Cai M. Mit1 transcription factor mediates methanol signaling and regulates the alcohol oxidase 1 (AOX1) promoter in Pichia pastoris.J. Biol. Chem. 2016; 291: 6245-6261Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 9Kumar N.V. Rangarajan P.N. The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris.J. Biol. Chem. 2012; 287: 34465-34473Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 10Shi L. Wang X. Wang J. Zhang P. Qi F. Cai M. Zhang Y. Zhou X. Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris.Genes Genomics. 2018; 40: 399-412Crossref PubMed Scopus (10) Google Scholar, 11Lee S.B. Kang H.S. Kim T.S. Nrg1 functions as a global transcriptional repressor of glucose-repressed genes through its direct binding to the specific promoter regions.Biochem. Biophys. Res. Commun. 2013; 439: 501-505Crossref PubMed Scopus (10) Google Scholar). Mxr1p regulates the expression of genes of acetate and amino acid metabolism as well and thus functions as a global regulator of central carbon metabolism (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Sahu U. Rangarajan P.N. 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.J. Biol. Chem. 2016; 291: 20588-20601Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). While Mxr1p primarily functions as a transcriptional activator, it is known to repress the expression of GT1 encoding glycerol transporter during glycerol metabolism (12Zhan C. Yang Y. Zhang Z. Li X. Liu X. Bai Z. Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris.FEMS Yeast Res. 2017; 17: 1-10Crossref Scopus (11) Google Scholar). Transcriptional activators bind to DNA through their DNA-binding domain (DBD) and activate transcription by interacting with proteins of preinitiation complex, either directly or via coactivators through their transactivation domains (TADs) (13Yang V.W. Eukaryotic transcription factors : Identification, characterization.J. Nutr. 1998; 128: 2045-2051Crossref PubMed Scopus (31) Google Scholar, 14Mitsis T. Efthimiadou A. Bacopoulou F. Vlachakis D. Chrousos G. Eliopoulos E. Transcription factors and evolution: An integral part of gene expression (review).World Acad. Sci. J. 2020; 2: 3-8Google Scholar). DBDs consist of motifs, such as zinc fingers, helix–turn–helix motif, or basic helix–loop–helix leucine zipper, whereas the TADs consist of amphipathic alpha helices, glutamine-rich domains, or proline-rich domains (15Arnold C.D. Nemčko F. Woodfin A.R. Wienerroither S. Vlasova A. Schleiffer A. Pagani M. Rath M. Stark A. A high-throughput method to identify trans-activation domains within transcription factor sequences.EMBO J. 2018; 37: 1-13Crossref PubMed Scopus (21) Google Scholar). Nuclear localization of transcription factors is facilitated by nuclear localization signals (NLSs), and the classical NLS is characterized by the presence of monopartite or bipartite NLSs consisting of one or two clusters of basic amino acids separated by a spacer, respectively (16Soniat M. Chook Y.M. Nuclear localization signals for four distinct Karyopherin-β nuclear import systems.Biochem. J. 2015; 468: 353-362Crossref PubMed Scopus (99) Google Scholar). P. pastoris Mxr1p possesses a DBD in the amino terminus consisting of two C2H2 zinc fingers, which exhibit strong homology to the DBD of S. cerevisiae Adr1p (6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar, 17Denis C.L. Young E.T. Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.Mol. Cell. Biol. 1983; 3: 360-370Crossref PubMed Scopus (81) Google Scholar, 18Kranthi B.V. Kumar R. Kumar N.V. Rao D.N. Rangarajan P.N. Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.Biochim. Biophys. Acta. 2009; 1789: 460-468Crossref PubMed Scopus (38) Google Scholar). Mxr1p DBD binds to Mxr1p response elements (MXREs) in the promoters of target genes bearing the consensus sequence 5′-CYCCNY-3′ (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Sahu U. Rangarajan P.N. 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.J. Biol. Chem. 2016; 291: 20588-20601Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 18Kranthi B.V. Kumar R. Kumar N.V. Rao D.N. Rangarajan P.N. Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.Biochim. Biophys. Acta. 2009; 1789: 460-468Crossref PubMed Scopus (38) Google Scholar, 19Kranthi B.V. Kumar H.R.V. Rangarajan P.N. Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.Yeast. 2010; 27: 705-711Crossref PubMed Scopus (26) Google Scholar). A TAD enriched in phenylalanine residues present between amino acid residues 246 and 280 of Mxr1p was shown to be functional during methanol metabolism but not ethanol metabolism (20Parua P.K. Ryan P.M. Trang K. Young E.T. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction.Mol. Microbiol. 2012; 85: 282-298Crossref PubMed Scopus (37) Google Scholar). Serine 215 residue located upstream of this TAD is phosphorylated in cells metabolizing ethanol but not methanol, and interaction of this phosphoserine with 14-3-3 protein inhibits the function of the N-terminal TAD (20Parua P.K. Ryan P.M. Trang K. Young E.T. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction.Mol. Microbiol. 2012; 85: 282-298Crossref PubMed Scopus (37) Google Scholar). Mxr1p is cytosolic in cells metabolizing glucose and localizes to the nucleus when cultured in media containing nonfermentable carbon sources (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar). Thus far, the NLS of Mxr1p has not been characterized. In S. cerevisiae, ethanol is metabolized into acetaldehyde, acetate, and acetyl-CoA by the sequential action of alcohol dehydrogenase, aldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS), respectively. In S. cerevisiae as well as Kluyveromyces lactis, expression of alcohol dehydrogenase 2 is regulated by Adr1p, which is regarded as the homolog of Mxr1p (17Denis C.L. Young E.T. Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.Mol. Cell. Biol. 1983; 3: 360-370Crossref PubMed Scopus (81) Google Scholar, 21Cardarelli S. D'Amici S. Tassone P. Tramonti A. Uccelletti D. Mancini P. Saliola M. Characterization of the transcription factor encoding gene, KlADR1: Metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.Microbiology (Reading). 2016; 162: 1933-1944Crossref PubMed Scopus (2) Google Scholar). However, the ability of P. pastoris Mxr1p to regulate genes of ethanol metabolism is not known. Considering the existing gaps, the present study was initiated to investigate the regulatory role of Mxr1p during ethanol metabolism, identify the NLS, and characterize the transactivation functions. Using high-throughput genome-wide RNA-Seq, we have identified novel target genes of Mxr1p essential for ethanol metabolism. Among these, ALD6-1 encoding an ALD, which is downregulated in Δmxr1, was further characterized. We have identified an NLS and a nine-amino acid TAD (9aaTAD) in the amino terminal region of Mxr1p required for the regulation of genes of ethanol metabolism. Finally, we have identified several novel target genes of Mxr1p required for methanol metabolism using DNA microarray. P. pastoris strains used in this study are listed in Table 1. To examine the role of Mxr1p in the regulation of genes of ethanol metabolism, GS115 and Δmxr1 were cultured in a medium containing yeast nitrogen base (YNB) and 1% ethanol (YNBE), and their growth pattern was studied. Growth of Δmxr1 was significantly retarded in comparison with that of GS115 (Fig. 1A) suggesting that Mxr1p regulates the expression of key genes of ethanol metabolism required for normal growth of P. pastoris. To gain insights into the mechanism of transcriptional regulation of genes of ethanol metabolism by Mxr1p, genome-wide RNA-Seq was carried out. GS115 and Δmxr1 were cultured in YNBE, RNA was isolated, and biological replicates were subjected to RNA-Seq. The datasets have been deposited in the Gene Expression Omnibus (GEO) database under accession identification number GSE168677. A total of 25 to 30 million reads were obtained for each sample after quality trimming. Samples were aligned to the K. phaffii strain CBS 7435 reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_900235035.1/). Differentially expressed genes were identified by normalizing the read counts in Δmxr1 to that of GS115 (control). Deletion of MXR1 resulted in significant changes in the transcriptome. Of 5424 genes, 170 were downregulated and 28 were upregulated in Δmxr1 (Supporting information 2). For this analysis, the threshold for statistical significance was considered an adjusted p value of <0.05 and log2 fold change of ±1 for upregulated and downregulated genes. The overall change and the most highly upregulated and downregulated genes are shown in the volcano plot and heat maps, respectively (Fig. 1, B–D). ALD6-1, encoding an ALD, was the most downregulated gene in Δmxr1 (greater than fivefold). Other genes downregulated in Δmxr1 include those encoding peroxisomal proteins, PP7435_Chr3-0349 encoding a transcription factor homologous to S. cerevisiae YLL054C (https://www.yeastgenome.org/locus/S000003977), PP7435_Chr2-0527 encoding glutathione transferase involved in detoxification of hydrogen peroxide, lipid metabolism genes (POX1, FAA2, and PCD1 encoding acyl Co-A oxidase, long-chain fatty acyl CoA synthetase, and pyrophosphatase, respectively) and GTH1 encoding a high-affinity glucose transporter. Interestingly, ADY2-3 encoding exporter of ammonia was downregulated, whereas MEP2 encoding ammonia transporter was upregulated in Δmxr1. CRC1, CAT2, and YAT1 encoding proteins involved in carnitine transport were highly upregulated in Δmxr1. Genes encoding acetyl CoA metabolizing enzymes, such as isocitrate lyase, isocitrate dehydrogenase, and isocitrate/isopropylmalate dehydrogenase, were also upregulated in Δmxr1 probably to facilitate metabolism of acetyl CoA via tricarboxylic acid cycle to generate energy in the absence of ALD6-1, the ethanol-metabolizing gene.Table 1Pichia pastoris strains used in this studyStrainDescriptionReferencesGS115his4(6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar)Δmxr1GS115, Ppmxr1Δ::Zeor(6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar)GS115-AGS115, HIS4::(PAPpALD6-1-Myc)This studyΔmxr1-AΔmxr1, HIS4::(PAPpALD6-1-Myc)This studyGS115-PA-GFPGS115, HIS4::(PA-GFP)This studyΔmxr1-PA-GFPΔmxr1, HIS4::(PA-GFP)This studyΔald6-1GS115, Ppald6-1Δ::ZeorThis studyΔald6-1-AΔald6-1, HIS4::(PAPpALD6-1-Myc)This studyΔmxr1-A(OE)Δmxr1, HIS4::(PGAPDHPpALD6-1-Myc)This studyGS115-PA-M1-GFPGS115, HIS4::(PA-M1-GFP)This studyGS115-PA-M2-GFPGS115, HIS4::(PA-M2-GFP)This studyGS115-PA-M3-GFPGS115, HIS4::(PA-M3-GFP)This studyGS115-PA-M4-GFPGS115, HIS4::(PA-M4-GFP)This studyΔmxr1-FLΔmxr1, Blar(PGAPDHPpMXR1-Myc)(4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar)Δmxr1-N400Δmxr1, Blar(PGAPDHPpMXR1N400-Myc)(4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar)Δmxr1-A-N400Δmxr1-A, Blar(PGAPDHPpMXR1N400-Myc)This studyΔmxr1-A-N400F∗Δmxr1-A, Hygr(PGAPDH-PpMXR1N400F∗-Myc)This studyΔmxr1-A-N400Q∗Δmxr1-A, Hygr(PGAPDH-PpMXR1N400Q∗-Myc)This studyΔmxr1-A-N400F∗Q∗Δmxr1-A, Hygr(PGAPDH-PpMXR1N400F∗Q∗-Myc)This studyΔmxr1-A-N250Δmxr1-A, Hygr(PGAPDHGFP-PpMXR1N250)This studyΔmxr1-A-N150Δmxr1-A, Hygr(PGAPDHGFP-PpMXR1N150)This studyΔmxr1-N250Δmxr1, Hygr(PGAPDHGFP-PpMXR1N250)This studyΔmxr1-N400ΔTADΔmxr1, Hygr(PGAPDHPpMXR1N400ΔTAD-Myc)This studyΔmxr1-N62Δmxr1, Hygr(PGAPDHGFP-PpMXR1N62)This studyΔmxr1-N81Δmxr1, Hygr(PGAPDHGFP-PpMXR1N81)This studyΔmxr1-N109Δmxr1, Hygr(PGAPDHGFP-PpMXR1N109)This studyΔmxr1-N250-M2Δmxr1, Hygr(PGAPDHGFP-PpMXR1N250-M2)This studyΔmxr1-N250-M1Δmxr1, Hygr(PGAPDHGFP-PpMXR1N250-M1)This study Open table in a new tab In S. cerevisiae, ALDs catalyze the conversion of acetaldehyde to acetate, the second step of ethanol metabolism (Fig. 1E). Since ALD6-1 is the most downregulated gene in Δmxr1 cultured in YNBE, we investigated its function and regulation in detail. P. pastoris genome harbors four ALD genes annotated as ALD6-1, ALD6-2, ALD4, and ALD2 (Table 2). Quantitation of the four ALD mRNAs by quantitative PCR (qPCR) indicated that ALD6-1 alone was downregulated in Δmxr1 during ethanol metabolism (Fig. 1F). Since ACS1 encoding ACS1 was shown to be downregulated in Δmxr1 during acetate metabolism (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), we examined ACS1 transcript levels in cells metabolizing ethanol. The results indicate that ACS1 mRNA levels are comparable in GS115 and Δmxr1 during ethanol metabolism (Fig. 1F) suggesting that Mxr1p does not regulate ACS1 expression during ethanol metabolism. It is pertinent to mention that P. pastoris homologs of transcription factor Cat8 regulate the expression of ACS1 during ethanol metabolism (22Barbay D. Mačáková M. Sützl L. De S. Mattanovich D. Gasser B. Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii.Curr. Genet. 2021; 67: 641-661Crossref PubMed Scopus (6) Google Scholar). 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. The results indicate that ALD6-1Myc is downregulated in Δmxr1-A (Fig. 1G). The gene encoding GFP was cloned downstream of 1.0 kb of ALD6-1 promoter (PA-GFP), and protein levels in the lysates of GS115 and Δmxr1 cultured in YNBE were examined by Western blotting using anti-GFP antibodies. GFP levels are also downregulated in Δmxr1 (Fig. 1H) suggesting that Mxr1p is likely to regulate ALD6-1 expression at the transcriptional level. To understand the importance of ALD6-1 in ethanol metabolism, gene encoding ALD6-1 was deleted to generate Δald6-1 (Fig. 1I), which exhibits retarded growth when cultured in YNBE (Fig. 1J). Expression of ALD6-1Myc from its own promoter in Δald6-1 (Fig. 1K) readily restores growth in YNBE (Fig. 1L). However, expression of ALD6-1Myc from GAPDH promoter in Δmxr1 (Fig. 1M) does not result in the restoration of growth in YNBE (Fig. 1N). Thus, ALD6-1 has a unique, essential, and nonredundant function during ethanol metabolism. The ability of ALD6-1 to restore growth of Δald6-1 but not Δmxr1 suggests that Mxr1p-regulated genes other than ALD6-1 (Fig. 1, C and D) are essential for ethanol metabolism.Table 2Pichia pastoris ALDsGeneP. pastoris CBS7435UniProt IDP. pastoris GS115UniProt IDALD6-1PP7435_Chr4-0972F2R0E4PAS_chr4_0043C4R6P6ALD6-2PP7435_Chr3-0183F2QUS7PAS_chr3_0987C4R664ALD4PP7435_Chr2-0787F2QSU4PAS_chr2-1_0853C4R0W4ALD2PP7435_Chr2-0843F2QSZ7PAS_chr2-1_0453C4R0Q6 Open table in a new tab Since Mxr1p regulates the transcription of target genes by binding to MXREs in their promoters bearing the consensus sequence 5′-CYCCNY-3′ (4Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl Co-A synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Sahu U. Rangarajan P.N. 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.J. Biol. Chem. 2016; 291: 20588-20601Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), we analyzed 1.0 kb of ALD6-1 promoter (PA) and identified three putative MXREs designated as MXRE1, MXRE2, and MXRE3 (Fig. 2A). Since point mutations within the 5′-CYCCNY-3′ motif of AOX1 MXREs abrogate Mxr1p binding (19Kranthi B.V. Kumar H.R.V. Rangarajan P.N. Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.Yeast. 2010; 27: 705-711Crossref PubMed Scopus (26) Google Scholar), 5′-CYCC-3′ sequence was mutated to 5′-CYCA-3′ in the PAMXREs (Fig. 2B). Oligonucleotides carrying these mutations were synthesized and designated as MXRE1-M, MXRE2-M, and MXRE3-M (Fig. 2B). A recombinant Mxr1p containing 150 N-terminal amino acids including the DBD, which specifically binds to MXREs of AOX1 promoter, was purified from Escherichia coli cell lysates (19Kranthi B.V. Kumar H.R.V. Rangarajan P.N. Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.Yeast. 2010; 27: 705-711Crossref PubMed Scopus (26) Google Scholar) and used in an electrophoretic mobility shift assay together with radiolabeled PAMXREs. The results indicate that Mxr1pN150 binds to wildtype but not the mutant PAMXREs (Fig. 2C). PA-GFP constructs carrying a point mutation in one or all three MXREs were generated (Fig. 2D), transformed into GS115, and GFP levels were examined by Western blotting using anti-GFP antibodies in the lysates of cells cultured in YNBE. The results indicate that mutation of MXRE1, MXRE2, or MXRE3 alone has no significant effect on GFP expression (Fig. 2E). However, GFP expression is significantly reduced when all the three are mutated (Fig. 2F) suggesting that a combination of two MXREs is sufficient for transactivation by Mxr1p from PA during ethanol metabolism. Key results obtained thus far are summarized in Figure 2G. Among the various P. pastoris transcription factors identified thus far (6Lin-Cereghino G.P. Godfrey L. de la Cruz B.J. Johnson S. Khuongsathiene S. Tolstorukov I. Yan M. Lin-Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.Mol. Cell. Biol. 2006; 26: 883-897Crossref PubMed Scopus (121) Google Scholar, 7Sahu U. Krishna Rao K. Rangarajan P.N. Trm1p, a Zn(II)2Cys6-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.Biochem. Biophys. Res. Commun. 2014; 451: 158-164Crossref PubMed Scopus (34) Google Scholar, 8Wang X. Wang Q. Wang J. Bai P. 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