Issue 24, 2018

E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Abstract

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M–K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.

Graphical abstract: E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Associated articles

Supplementary files

Article information

Article type
Edge Article
Submitted
30 Jan 2018
Accepted
22 May 2018
First published
24 May 2018
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2018,9, 5383-5388

E. coli surface display of streptavidin for directed evolution of an allylic deallylase

T. Heinisch, F. Schwizer, B. Garabedian, E. Csibra, M. Jeschek, J. Vallapurackal, V. B. Pinheiro, P. Marlière, S. Panke and T. R. Ward, Chem. Sci., 2018, 9, 5383 DOI: 10.1039/C8SC00484F

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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