DVOŘÁK, Pavel, Esteban MARTÍNEZ-GARCÍA and Victor DE LORENZO. Empowering Pseudomonas putida with surface-displayed designer protein scaffolds. In FEMS 2019: 8th Congress of European Microbiologists. 2019.
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Basic information
Original name Empowering Pseudomonas putida with surface-displayed designer protein scaffolds
Name in Czech Konstrukce bakterie Pseudomonas putida se syntetickými proteinovými lešeními na povrchu
Authors DVOŘÁK, Pavel, Esteban MARTÍNEZ-GARCÍA and Victor DE LORENZO.
Edition FEMS 2019: 8th Congress of European Microbiologists, 2019.
Other information
Original language English
Type of outcome Presentations at conferences
Country of publisher Czech Republic
Confidentiality degree is not subject to a state or trade secret
WWW URL
Organization unit Faculty of Science
Keywords (in Czech) Pseudomonas putida, syntetická biologie, celulozomy
Keywords in English Pseudomonas putida, cellulosomes, synthetic biology, surface display
Tags International impact
Changed by Changed by: doc. Mgr. Pavel Dvořák, Ph.D., učo 151419. Changed: 11/2/2020 15:00.
Abstract
Pseudomonas putida KT2440, the best-characterized and safe pseudomonad, belongs among the most promising bacterial hosts for synthetic biology and biotechnology endeavours. P. putida-based applications would greatly benefit from novel catalytic activities displayed on its surface. However, recombinant protein secretion in this G- host is inefficient and not well established. We sought to meet this challenge by developing a system for efficient display of recombinant proteins on P. putida surface by employing a synthetic biology approach. New engineered P. putida strains EM42 and EM371, with reduced genomes and altered physiological properties, were combined with surface display of small anchoring protein scaffolds inspired by natural cellulosomes. These synthetic scaffolds served as docks for recombinant proteins tagged with complementary binding domains. Attachment of the proteins to the P. putida surface was verified by enzyme assays, confocal microscopy, fluorescence spectroscopy, and flow cytometry. Synthetic scaffolds containing one or two cohesin binding domains were successfully displayed on the surface of both P. putida strains with one of four tested autotransporter systems. Beta-glucosidase and two different fluorescent proteins were anchored to the surface of P. putida EM42 and EM371 with high efficiency especially in the latter case. This study shows the benefits of the strain EM371 for the surface display of recombinant proteins and introduces designer cellulosome strategy tailored for P. putida.
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