fbdd96b81bb26fd9875abc98726682fd catalysts_a2021v11n10p1192.pdf 168b029e6e27360e846441c1e8a0fbaf31b22498 catalysts_a2021v11n10p1192.pdf 898127f43db89bd0f671774fcd4f0afa3d5b24252da6228f47d4de07c424abea catalysts_a2021v11n10p1192.pdf Title: Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability Subject: Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of theK. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel). Keywords: Rhizopus oryzae lipase; Komagataella phaffii; prosequence; flavor; biodiesel; biocatalysis Author: Josu López-Fernández, Maria Dolors Benaiges and Francisco Valero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Oct 20 07:40:46 2021 CEST ModDate: Wed Oct 20 07:43:29 2021 CEST 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: 922195 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- UNTTAD+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 QKHJEW+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 VWQFPP+URWPalladioL-BoldItal Type 1 Custom yes yes no 19 0 IGPRAW+URWPalladioL-Roma Type 1 Custom yes yes no 23 0 ENXZUH+VnURWPalladioL-Bold Type 1 Custom yes yes no 27 0 HCRKXL+VnURWPalladioL Type 1 Custom yes yes no 32 0 CGDLTM+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 40 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 62 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 66 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 70 0 XYPWHM+CMSY10 Type 1 Builtin yes yes no 82 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 89 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 96 0 GKEJOP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 110 0 GKEJPA+PalatinoLinotype CID TrueType Identity-H yes yes yes 113 0 GKEJPB+PalatinoLinotype TrueType WinAnsi yes yes no 119 0 GKEJPD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 122 0 GKEJOP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 136 0 GKEJPB+PalatinoLinotype TrueType WinAnsi yes yes no 139 0 GKEJPD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 142 0 GKEJOP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 159 0 GKEJPB+PalatinoLinotype TrueType WinAnsi yes yes no 162 0 GKEJPD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 165 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:28:02 CEST RepresentationInformation: catalysts_a2021v11n10p1192.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:58:30 CEST Size: 922195 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 422 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Batch and Fed-Batch Bioprocess Strategies. Lipase Production under Inducible PAOX1 and Constitutive PGAP Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: N-Terminal Amino Acids in proROL, rROL and proROLm Free Enzymes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Stability of EB-proROL and EB-rROL in the Presence of Organic Solvents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Transesterification and Esterification Reactions. Initial Reaction Rate and Operational Stability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Biodiesel Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Ethyl Butyrate Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Chemicals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Strains and Plasmids Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Batch Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Fed-Batch Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Lipase Production and Activity Assessment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: N-Terminal Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Lipase Immobilization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Transesterification and Esterification Reactions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Gas Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Immobilized Lipase Stability against Organic Solvents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability Author: Josu López-Fernández, Maria Dolors Benaiges and Francisco Valero Subject: Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of theK. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel). 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