Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by various bacteria under nutrient-limiting conditions with excess carbon. These polymers accumulate as intracellular granules coated with granule-associated proteins (GAPs), including phasins, the most abundant and functionally d...

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Autores: Manoli, Maria-Tsampika, Llamas, Paula, Blanco Parte, Francisco German, Hernández-Arriaga, A.M., Torices, Maria Isabel, Prieto, María Auxiliadora
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2026
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/419455
Acceso en línea:http://hdl.handle.net/10261/419455
https://api.elsevier.com/content/abstract/scopus_id/105027917114
Access Level:acceso abierto
Palabra clave:Phasins
Polyhydroxyalkanoates
Pseudomonas putida
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dc.title.none.fl_str_mv Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
title Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
spellingShingle Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
Manoli, Maria-Tsampika
Phasins
Polyhydroxyalkanoates
Pseudomonas putida
title_short Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
title_full Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
title_fullStr Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
title_full_unstemmed Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
title_sort Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
dc.creator.none.fl_str_mv Manoli, Maria-Tsampika
Llamas, Paula
Blanco Parte, Francisco German
Hernández-Arriaga, A.M.
Torices, Maria Isabel
Prieto, María Auxiliadora
author Manoli, Maria-Tsampika
author_facet Manoli, Maria-Tsampika
Llamas, Paula
Blanco Parte, Francisco German
Hernández-Arriaga, A.M.
Torices, Maria Isabel
Prieto, María Auxiliadora
author_role author
author2 Llamas, Paula
Blanco Parte, Francisco German
Hernández-Arriaga, A.M.
Torices, Maria Isabel
Prieto, María Auxiliadora
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv European Commission
CSIC - Plataforma Temática Interdisciplinar del CSIC plásticos Sostenibles (PTI SusPlast)
Ministerio de Ciencia, Innovación y Universidades (España)
Manoli, Maria-Tsampika [0000-0002-3075-4634]
Prieto, María Auxiliadora [0000-0002-8038-1223]
Manoli, Maria-Tsampika [0000-0002-3075-4634]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Phasins
Polyhydroxyalkanoates
Pseudomonas putida
topic Phasins
Polyhydroxyalkanoates
Pseudomonas putida
description Polyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by various bacteria under nutrient-limiting conditions with excess carbon. These polymers accumulate as intracellular granules coated with granule-associated proteins (GAPs), including phasins, the most abundant and functionally diverse GAPs. Phasins play essential roles in granule formation, PHA metabolism, and have emerged as promising tools for functionalizing PHA surfaces in applications such as enzyme immobilization and drug delivery. In this study, we assessed the binding affinity and influence of two phasins, PhaP1 and PhaI, on PHA accumulation and granule morphology in three engineered Pseudomonas putida KT2440 strains (PP00_01, PP01_02, and PP05_17), optimized for tailored PHA production with distinct monomer compositions (medium-, short-chain-length PHAs, and the hybrid scl/mcl-PHA producer, respectively). Phasin-msfGFP fusion proteins were expressed and monitored using in vivo fluorescence binding assays. PHA accumulation was evaluated via microscopy and GC-MS. Despite similar PHA yields (%PHA/CDW) in strains with and without phasins, PhaI expression in mcl-PHA-producing strain PP00_01 led to increased granule number and homogeneity, confirmed by transmission electron microscopy. Additionally, flow cytometry analyses revealed differential surface affinities of phasins depending on polymer type, indicating that both phasin type and PHA physicochemical properties influence binding strength. PhaP1 and PhaI displayed distinct preferences for scl- and mcl PHAs, respectively. Overall, this work underscores the dual role of phasins in modulating PHA production and enabling selective functionalization of PHA granules, supporting their use as bio-affinity tags for polymer-based biotechnological applications.
publishDate 2026
dc.date.none.fl_str_mv 2026
2026
2026
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url http://hdl.handle.net/10261/419455
https://api.elsevier.com/content/abstract/scopus_id/105027917114
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spelling Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assaysManoli, Maria-TsampikaLlamas, PaulaBlanco Parte, Francisco GermanHernández-Arriaga, A.M.Torices, Maria IsabelPrieto, María AuxiliadoraPhasinsPolyhydroxyalkanoatesPseudomonas putidaPolyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by various bacteria under nutrient-limiting conditions with excess carbon. These polymers accumulate as intracellular granules coated with granule-associated proteins (GAPs), including phasins, the most abundant and functionally diverse GAPs. Phasins play essential roles in granule formation, PHA metabolism, and have emerged as promising tools for functionalizing PHA surfaces in applications such as enzyme immobilization and drug delivery. In this study, we assessed the binding affinity and influence of two phasins, PhaP1 and PhaI, on PHA accumulation and granule morphology in three engineered Pseudomonas putida KT2440 strains (PP00_01, PP01_02, and PP05_17), optimized for tailored PHA production with distinct monomer compositions (medium-, short-chain-length PHAs, and the hybrid scl/mcl-PHA producer, respectively). Phasin-msfGFP fusion proteins were expressed and monitored using in vivo fluorescence binding assays. PHA accumulation was evaluated via microscopy and GC-MS. Despite similar PHA yields (%PHA/CDW) in strains with and without phasins, PhaI expression in mcl-PHA-producing strain PP00_01 led to increased granule number and homogeneity, confirmed by transmission electron microscopy. Additionally, flow cytometry analyses revealed differential surface affinities of phasins depending on polymer type, indicating that both phasin type and PHA physicochemical properties influence binding strength. PhaP1 and PhaI displayed distinct preferences for scl- and mcl PHAs, respectively. Overall, this work underscores the dual role of phasins in modulating PHA production and enabling selective functionalization of PHA granules, supporting their use as bio-affinity tags for polymer-based biotechnological applications.This research received funding from the European Union's Horizon 2020 research and innovation program under grant agreement numbers 814418 (SinFonia), 870294 (MIX-up), and 101081782 (DeCypher). This work was supported by the CSIC Interdisciplinary Thematic Platform (PTI+) Sustainable Plastics towards a Circular Economy (PTI-Susplast), OPENVIRO (PID2023-146557OB-C21), NOVELPOL (202320E107), PID2022-139209OB-C21 funded by MICIU/AEI/10.13039/501100011033 (Spain) and ERDF (A way of making Europe). We thank the CIB scientific facilities (gas chromatography, flow cytometry, and transmission electron microscopy). We are grateful to Dr. Beatriz Maestro, and Dr. Jesús Miguel Sanz Morales for insightful discussions, to Dr. Manuel Santiago Godoy for his assistance in pBDN and pBDN2 plasmid construction, and to Rodrigo Sanchez Tarjuelo for assistance with flow cytometry assays. We also acknowledge the technical support of Ana Valencia and Melissa Méndez González.Peer reviewedElsevierEuropean CommissionCSIC - Plataforma Temática Interdisciplinar del CSIC plásticos Sostenibles (PTI SusPlast)Ministerio de Ciencia, Innovación y Universidades (España)Manoli, Maria-Tsampika [0000-0002-3075-4634]Prieto, María Auxiliadora [0000-0002-8038-1223]Manoli, Maria-Tsampika [0000-0002-3075-4634]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202620262026info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/419455https://api.elsevier.com/content/abstract/scopus_id/105027917114reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#EC/H2020/814418info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2023-146557OB-C21202320E107info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2022-139209OB-C21https://doi.org/10.1016/j.ijbiomac.2026.150332Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4194552026-05-22T06:33:51Z
score 15,81155

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