Publication details

Empowering Pseudomonas putida with surface-displayed designer protein scaffolds



Year of publication 2019
Type Appeared in Conference without Proceedings
MU Faculty or unit

Faculty of Science

Description 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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