Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior.

Martin Gustavsson,Susanna Lundh,David Hörnström,Gen Larsson,Jaroslav Belotserkovsky

doi:10.1038/srep36117

Abstract

Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.

Highlights

Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications
Growing environmental concern requires the design of sustainable processes to replace products that are currently derived from fossil sources and has promoted the development of new bioremediation methods to handle the continued pollution of air, soil and water
Continued research advances allow the directed evolution of both cells and enzymes and a successful example of transplantation of a complete heterologous pathway is the introduction of Saccharomyces and Klebsiella enzymes into Escherichia coli for the production of 1,3-propanediol[1]

Summary

Introduction

It is considered state-of-the-art to engineer living organisms for various biotechnology applications. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin This resulted in a color change and creation of a black cell exterior. ATs offer an elegant solution to the biggest obstacle for Gram-negative surface display, namely translocation through the outer membrane (OM) in the absence of adenosine triphosphate and without assistance of a proton motive force This obstacle is overcome by the expression of a single polypeptide bearing all the required functionalities: an N-terminal signal peptide directing the protein to the periplasm, a “passenger” domain responsible for the functionality of each individual AT, and a C-terminal β-barrel that acts as an OM anchor and as a pore for passenger domain transport[7]. The positioning of enzymes on the surface by use of appropriate mechanisms for expression and translocation to the surface constitutes a new cell modification strategy, highlighting an example of process facilitation www.nature.com/scientificreports/

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