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Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of Pseudomonas putida

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    Research Article

    Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of Pseudomonas putida
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2020, 9, 10, 2749–2764
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    https://doi.org/10.1021/acssynbio.0c00276
    Published September 2, 2020
    Copyright © 2020 American Chemical Society
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    Abstract

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    The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor—a display of designer enzyme-anchoring protein “scaffoldins”, encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.

    Copyright © 2020 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.0c00276.

    • Discussions of effect of scaffoldin and autotransporter expression on the viability of P. putida cells, SDS-PAGE and Western blot analyses, and dot blot analysis, tables of strains, plasmids, and oligonucleotide primers used, and figures of schematic reconstruction, effect of scaf19L and scaf19LKT expression, Western blot analysis, SDS-polyacrylamide gels, effect of an autotransporter expression, and supporting sequences (PDF)

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    This article is cited by 19 publications.

    1. Siddhant Gulati, Qing Sun. Complete Enzymatic Depolymerization of Polyethylene Terephthalate (PET) Plastic Using a Saccharomyces cerevisiae-Based Whole-Cell Biocatalyst. Environmental Science & Technology Letters 2025, 12 (4) , 419-424. https://doi.org/10.1021/acs.estlett.5c00190
    2. Zijie Li, Wanjie Li, Yasen Wang, Zhou Chen, Hideki Nakanishi, Xiangyang Xu, Xiao-Dong Gao. Establishment of a Novel Cell Surface Display Platform Based on Natural “Chitosan Beads” of Yeast Spores. Journal of Agricultural and Food Chemistry 2022, 70 (24) , 7479-7489. https://doi.org/10.1021/acs.jafc.2c01983
    3. Miguel Silva, Stefano Donati, Pavel Dvořák. Advances in engineering substrate scope of Pseudomonas cell factories. Current Opinion in Biotechnology 2025, 92 , 103270. https://doi.org/10.1016/j.copbio.2025.103270
    4. Haixia Wang, Jiahong Zhu, Meng Sun, Mengjie Gu, Xiya Xie, Tongtong Ying, Zeling Zhang, Weihong Zhong. Biodegradation of combined pollutants of polyethylene terephthalate and phthalate esters by esterase-integrated Pseudomonas sp. JY-Q with surface-co-displayed PETase and MHETase. Synthetic and Systems Biotechnology 2025, 10 (1) , 10-22. https://doi.org/10.1016/j.synbio.2024.08.001
    5. Victor de Lorenzo, Danilo Pérez-Pantoja, Pablo I. Nikel, . Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis. Journal of Bacteriology 2024, 206 (7) https://doi.org/10.1128/jb.00136-24
    6. Esteban Martínez-García, Víctor de Lorenzo. Pseudomonas putida as a synthetic biology chassis and a metabolic engineering platform. Current Opinion in Biotechnology 2024, 85 , 103025. https://doi.org/10.1016/j.copbio.2023.103025
    7. Helena Nevalainen, Shivam Aggarwal, Nidhi Adlakha. Sources, Properties, and Modification of Lignocellulolytic Enzymes for Biomass Degradation. 2024, 567-605. https://doi.org/10.1007/978-94-007-6308-1_23
    8. Caroline R. Amendola, William T. Cordell, Colin M. Kneucker, Caralyn J. Szostkiewicz, Morgan A. Ingraham, Michela Monninger, Rosemarie Wilton, Brian F. Pfleger, Davinia Salvachúa, Christopher W. Johnson, Gregg T. Beckham. Comparison of wild-type KT2440 and genome-reduced EM42 Pseudomonas putida strains for muconate production from aromatic compounds and glucose. Metabolic Engineering 2024, 81 , 88-99. https://doi.org/10.1016/j.ymben.2023.11.004
    9. Sarah Moraïs, Johanna Stern, Lior Artzi, Carlos M. G. A. Fontes, Edward A. Bayer, Itzhak Mizrahi. Carbohydrate Depolymerization by Intricate Cellulosomal Systems. 2023, 53-77. https://doi.org/10.1007/978-1-0716-3151-5_4
    10. Helena Nevalainen, Shivam Aggarwal, Nidhi Adlakha. Sources, Properties, and Modification of Lignocellulolytic Enzymes for Biomass Degradation. 2023, 1-39. https://doi.org/10.1007/978-94-007-6724-9_23-1
    11. Dalimil Bujdoš, Barbora Popelářová, Daniel C. Volke, Pablo I. Nikel, Nikolaus Sonnenschein, Pavel Dvořák. Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model. Metabolic Engineering 2023, 75 , 29-46. https://doi.org/10.1016/j.ymben.2022.10.011
    12. Shen-Long Tsai, Qing Sun, Wilfred Chen. Advances in consolidated bioprocessing using synthetic cellulosomes. Current Opinion in Biotechnology 2022, 78 , 102840. https://doi.org/10.1016/j.copbio.2022.102840
    13. Myeong-Eun Lee, Young Jin Ko, Dong-Hyeok Hwang, Byeong-Hyeon Cho, Wu-Young Jeong, Nisha Bhardwaj, Sung Ok Han. Surface display of enzyme complex on Corynebacterium glutamicum as a whole cell biocatalyst and its consolidated bioprocessing using fungal-pretreated lignocellulosic biomass. Bioresource Technology 2022, 362 , 127758. https://doi.org/10.1016/j.biortech.2022.127758
    14. Victor de Lorenzo, Esteban Martínez‐García, Tomás Aparicio. Microbial‐based Bioremediation at a Global Scale. 2022, 325-336. https://doi.org/10.1002/9781119762621.ch26
    15. Stanislav Obruča, Pavel Dvořák, Petr Sedláček, Martin Koller, Karel Sedlář, Iva Pernicová, David Šafránek. Polyhydroxyalkanoates synthesis by halophiles and thermophiles: towards sustainable production of microbial bioplastics. Biotechnology Advances 2022, 58 , 107906. https://doi.org/10.1016/j.biotechadv.2022.107906
    16. Kaitlin R. Clarke, Lilian Hor, Akila Pilapitiya, Joen Luirink, Jason J. Paxman, Begoña Heras. Phylogenetic Classification and Functional Review of Autotransporters. Frontiers in Immunology 2022, 13 https://doi.org/10.3389/fimmu.2022.921272
    17. Ziyu Wang, Yifei Zheng, Mengke Ji, Xu Zhang, Huan Wang, Yuemeng Chen, Qiong Wu, Guo‐Qiang Chen. Hyperproduction of PHA copolymers containing high fractions of 4‐hydroxybutyrate (4HB) by outer membrane‐defected Halomonas bluephagenesis grown in bioreactors. Microbial Biotechnology 2022, 15 (5) , 1586-1597. https://doi.org/10.1111/1751-7915.13999
    18. Stanislav Juračka, Barbora Hrnčířová, Barbora Burýšková, Daniel Georgiev, Pavel Dvořák. Building the SynBio community in the Czech Republic from the bottom up: You get what you give. Biotechnology Notes 2022, 3 , 124-134. https://doi.org/10.1016/j.biotno.2022.11.002
    19. Ziyu Wang, Qin Qin, Yifei Zheng, Fajin Li, Yiqing Zhao, Guo-Qiang Chen. Engineering the permeability of Halomonas bluephagenesis enhanced its chassis properties. Metabolic Engineering 2021, 67 , 53-66. https://doi.org/10.1016/j.ymben.2021.05.010
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2020, 9, 10, 2749–2764
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.0c00276
    Published September 2, 2020
    Copyright © 2020 American Chemical Society
    Request reuse permissions

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