615cd25ffa89a41dfa41ec70d42b3de7 pmc_11767246.pdf 855ed67b867c8216e5e1e3bd31d13643f84476f0 pmc_11767246.pdf 6ecca90eda529ff045b3f0d05141c4bec6c134e9d5f9b0dff027743964476976 pmc_11767246.pdf Title: A Rewired NADPH-Dependent Redox Shuttle for Testing Peroxisomal Compartmentalization of Synthetic Metabolic Pathways in Komagataella phaffii Subject: The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors—acetyl-CoA, NADPH, and ATP—with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast Komagataella phaffii. To this end, the malonyl-CoA reductase pathway for 3-HP production was targeted into the peroxisome of K. phaffii using the PTS1-tagging system, and further tested with different carbon sources. Thereafter, we used compartmentalized 3-HP production as a reporter system to showcase the impact of different strategies aimed at enhancing the peroxisomal NADPH pool. Co-overexpression of genes encoding a NADPH-dependent redox shuttle from Saccharomyces cerevisiae (IDP2/IDP3) significantly increased 3-HP yields across all substrates, whereas peroxisomal targeting of the S. cerevisiae NADH kinase Pos5 failed to improve 3-HP production. This study highlights the potential of using peroxisomal 3-HP production as a biosensor for evaluating peroxisomal acetyl-CoA and NAPDH availability by simply quantifying 3-HP, demonstrating its potential for peroxisome-based metabolic engineering in yeast. Keywords: peroxisome; metabolic engineering; 3-hydroxypropionic acid; Pichia pastoris; Komagataella phaffii; acetyl-CoA; NADPH; redox shuttle Author: Albert Fina, Sílvia Àvila-Cabré, Enrique Vázquez-Pereira, Joan Albiol and Pau Ferrer Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Mon Dec 30 08:52:51 2024 CET ModDate: Mon Dec 30 09:01:09 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1599319 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- RTYZKO+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 YWRZJB+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 JCXQFW+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 MBKLTL+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 XDGMFC+URWPalladioL-BoldItal Type 1 Custom yes yes yes 32 0 AYVXZI+VnURWPalladioL Type 1 Custom yes yes yes 37 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 63 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes yes 71 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 79 0 DBOPMP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 97 0 DBOPNA+PalatinoLinotype CID TrueType Identity-H yes yes yes 100 0 DBOPOA+PalatinoLinotype TrueType WinAnsi yes yes no 106 0 DBOPOB+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 109 0 DBOPOC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 115 0 DBPAKB+PalatinoLinotype TrueType MacRoman yes yes no 118 0 DBOPMP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 132 0 DBOPNA+PalatinoLinotype CID TrueType Identity-H yes yes yes 135 0 DBOPOA+PalatinoLinotype TrueType WinAnsi yes yes no 141 0 DBOPOB+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 144 0 DBOPOC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 150 0 DBPAKB+PalatinoLinotype TrueType MacRoman yes yes no 153 0 DBOPMP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 170 0 DBOPNA+PalatinoLinotype CID TrueType Identity-H yes yes yes 173 0 DBOPOA+PalatinoLinotype TrueType WinAnsi yes yes no 179 0 DBOPOC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 182 0 DBPAKB+PalatinoLinotype TrueType MacRoman yes yes no 185 0 DBOPMP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 202 0 DBOPNA+PalatinoLinotype CID TrueType Identity-H yes yes yes 205 0 DBOPOA+PalatinoLinotype TrueType WinAnsi yes yes no 211 0 DBOPOB+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 214 0 DBOPOC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 220 0 DBPAKB+PalatinoLinotype TrueType MacRoman yes yes no 223 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-02-17 16:54:18 CET RepresentationInformation: pmc_11767246.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-02-12 02:32:59 CET Size: 1599319 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 468 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Construction of Strains Destination: subsection.2.1 Item: Title: Screening Conditions Destination: subsection.2.2 Item: Title: Analytical Methods Destination: subsection.2.3 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Construction of K. phaffii Strains Engineered for 3-HP Production in Peroxisomes Destination: subsection.3.1 Item: Title: Using 3-HP Produced from Oleic Acid as a Reporter Metabolite to Detect Peroxisomal NADPH Levels Destination: subsection.3.2 Item: Title: Testing the Potential of Peroxisomal 3-HP Production as a Synthetic Biosensor in K. phaffii Strains Growing on Different Carbon Sources Destination: subsection.3.3 Item: Title: Comparison of the Peroxisomal and Cytosolic Variants of the 3-HP Producing Strains Destination: subsection.3.4 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: appendix.A. Info: Title: A Rewired NADPH-Dependent Redox Shuttle for Testing Peroxisomal Compartmentalization of Synthetic Metabolic Pathways in Komagataella phaffii Author: Albert Fina, Sílvia Àvila-Cabré, Enrique Vázquez-Pereira, Joan Albiol and Pau Ferrer Subject: The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors acetyl-CoA, NADPH, and ATP with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast Komagataella phaffii. To this end, the malonyl-CoA reductase pathway for 3-HP production was targeted into the peroxisome of K. phaffii using the PTS1-tagging system, and further tested with different carbon sources. Thereafter, we used compartmentalized 3-HP production as a reporter system to showcase the impact of different strategies aimed at enhancing the peroxisomal NADPH pool. Co-overexpression of genes encoding a NADPH-dependent redox shuttle from Saccharomyces cerevisiae (IDP2/IDP3) significantly increased 3-HP yields across all substrates, whereas peroxisomal targeting of the S. cerevisiae NADH kinase Pos5 failed to improve 3-HP production. This study highlights the potential of using peroxisomal 3-HP production as a biosensor for evaluating peroxisomal acetyl-CoA and NAPDH availability by simply quantifying 3-HP, demonstrating its potential for peroxisome-based metabolic engineering in yeast. 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