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

Cite this: ACS Synth. Biol. 2020, 9, 10, 2749–2764
Publication Date (Web):September 2, 2020
https://doi.org/10.1021/acssynbio.0c00276
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.

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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 14 publications.

    1. 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
    2. 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
    3. 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
    4. 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
    5. 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
    6. 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
    7. 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
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    9. 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
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    11. 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
    12. 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
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    14. 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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