Abstract
Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.
Highlights
Engineering metabolic pathways into heterologous host cells to produce valuable chemical compounds and biofuels is a major goal of synthetic biology [1,2]
Using the Eut bacterial microcompartments (BMCs) shell proteins from S. enterica as our model system, we demonstrate that proteinaceous compartments can be engineered in E. coli
We show that heterologous proteins are efficiently targeted into the recombinant compartments; enabling engineering of multi-step biocatalysis within tailored microcompartments as in vivo or in vitro nano-bioreactors
Summary
Engineering metabolic pathways into heterologous host cells to produce valuable chemical compounds and biofuels is a major goal of synthetic biology [1,2]. Co-localization can be achieved, for example, by tethering enzymes to structures such as protein scaffolds or lipid membranes [4,5] Drawing upon this approach, a synthetic protein scaffold was recently shown to dramatically increase flux through an engineered biosynthetic pathway [6]. Compartmentalization allows more stringent control over substrate and product transport to and from enzyme assemblies. It protects the organism from harmful reaction intermediates. It is known that several bacteria form proteinaceous shells that encapsulate functionally related enzymes. These are collectively referred to as bacterial microcompartments (BMCs) [7]. Recent advances in our understanding of BMC structure and function open up possibilities for engineering them into nano-bioreactors for biosynthesis and biocatalysis
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