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Expanding the biocatalytic capabilities of Pseudomonas putida with surface-displayed designer protein scaffolds

Název česky Posílení katalytických schopností Pseudomonas putida pomocí syntetických proteinových lešení na povrchu buňky


Rok publikování 2019
Druh Další prezentace na konferencích
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis P. putida KT2440, the best-characterized, safe pseudomonad, belongs among the most pronounced bacterial workhorses in synthetic biology and biotechnology.1 It has been frequently used in biodegradation and bioremediation endeavours2 and recently is drawing a considerable attention also as a promising bacterial platform for valorization of lignocellulose-derived aromatic compounds and sugars.3,4 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 EM425 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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