f4085c23fe68fdc2b8e8e270f18a608d ijms-27-06561-v2.pdf df5c370733f0fb8f24545bd1c2ef5c59430cf83e ijms-27-06561-v2.pdf 79e06fc06916b0036bc763ab592b9f1787672553f91c271de3dbbe7fab2dd52a ijms-27-06561-v2.pdf Title: Komagataella phaffii Encodes Two Functional Pho4 Transcription Factors Subject: The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter, we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in Saccharomyces cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter, and secreted phosphatase activity. Therefore, both transcription factors display partially overlapping functions, respond differently to diverse stimuli, and together constitute a key component in the adaptation to multiple stresses. Keywords: transcriptional regulation; phosphate starvation; Pho4 transcription factors; Komagataella phaffii; Saccharomyces cerevisiae Author: Marcel Albacar, Asier González, Rui Wang, Antonio Casamayor and Joaquín Ariño Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Jul 24 10:57:01 2026 CEST ModDate: Fri Jul 24 11:27:32 2026 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 15855639 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IQESQO+URWPalladioL-Roma Type 1 Custom yes yes yes 136 0 NGOQYE+URWPalladioL-BoldItal Type 1 Custom yes yes yes 137 0 KYHUCB+URWPalladioL-Ital Type 1 Custom yes yes yes 138 0 QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 139 0 ZWMYRW+URWPalladioL-Bold Type 1 Custom yes yes yes 140 0 SWBBCH+CMSY10 Type 1 Builtin yes yes yes 196 0 ROWTUD+PazoMath Type 1 Builtin yes yes yes 237 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 255 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-10-02 04:05:02 CEST RepresentationInformation: ijms-27-06561-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-10-01 05:20:46 CEST Size: 15855639 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 731 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Identification of PHO4 Candidates in K. phaffii Item: Title: Phenotypic Analysis of Single and Double Pho4 K. phaffii Mutants Reveals Both Specialized and Redundant Functions Item: Title: The Role of Pho4(A) and Pho4(B) in the Transcriptional Response to Pi Starvation Item: Title: Pho4(A) Contributes to the Regulation of the Expression of PHO89 Under High pH Stress Item: Title: Both K. phaffii Pho4 Transcription Factors Are Functional in S. cerevisiae Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Growth Conditions and Recombinant DNA Techniques Item: Title: Generation of K. phaffii PHO4(A) and PHO4(B) Mutants Item: Title: RNA Preparation and Transcriptomic Analysis Item: Title: Phenotypic Assays Item: Title: Expression of GFP from the PHO89 Promoter in K. phaffii Item: Title: Determination of Pho84-GFP Expression by Flow-Cytometry Item: Title: Determination of Secreted Phosphatase Item: Title: Other Methods Item: Title: References Info: Title: Komagataella phaffii Encodes Two Functional Pho4 Transcription Factors Author: Marcel Albacar, Asier González, Rui Wang, Antonio Casamayor and Joaquín Ariño Subject: The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter, we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in Saccharomyces cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter, and secreted phosphatase activity. Therefore, both transcription factors display partially overlapping functions, respond differently to diverse stimuli, and together constitute a key component in the adaptation to multiple stresses. 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Subtype: Link Rect: 421, 27, 560, 37 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 478, 743, 515, 754 Annotation: Subtype: Link Rect: 521, 743, 557, 754 Annotation: Subtype: Link Rect: 341, 705, 379, 716 Annotation: Subtype: Link Rect: 385, 705, 421, 716 Annotation: Subtype: Link Rect: 236, 680, 273, 690 Annotation: Subtype: Link Rect: 280, 680, 316, 690 Annotation: Subtype: Link Rect: 140, 655, 177, 665 Annotation: Subtype: Link Rect: 183, 655, 219, 665 Annotation: Subtype: Link Rect: 327, 628, 364, 639 Annotation: Subtype: Link Rect: 370, 628, 406, 639 Annotation: Subtype: Link Rect: 274, 604, 311, 614 Annotation: Subtype: Link Rect: 317, 604, 353, 614 Annotation: Subtype: Link Rect: 335, 578, 372, 588 Annotation: Subtype: Link Rect: 378, 578, 414, 588 Annotation: Subtype: Link Rect: 385, 552, 422, 563 Annotation: Subtype: Link Rect: 429, 552, 465, 563 Annotation: Subtype: Link Rect: 263, 526, 301, 537 Annotation: Subtype: Link Rect: 307, 526, 343, 537 Annotation: Subtype: Link Rect: 384, 501, 421, 512 Annotation: Subtype: Link Rect: 428, 501, 464, 512 Annotation: Subtype: Link Rect: 117, 476, 155, 486 Annotation: Subtype: Link Rect: 161, 476, 197, 486 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Checksum: f33f492a Type: CRC32 Checksum: f4085c23fe68fdc2b8e8e270f18a608d Type: MD5 Checksum: df5c370733f0fb8f24545bd1c2ef5c59430cf83e Type: SHA-1 Checksum: 79e06fc06916b0036bc763ab592b9f1787672553f91c271de3dbbe7fab2dd52a Type: SHA-256