Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli

Held, Mark; Kolb, Alexander L.; Perdue, Sarah; Hsu, Szu Yi; Bloch, Sarah E.; Quin, Maureen B.

doi:10.1038/srep24359

Cited by 52 publications

(42 citation statements)

References 82 publications

(156 reference statements)

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“…Spatial organization of enzymes by encapsulation within protein shells (as exemplified in MCP systems) has the ability to accelerate catalysis, prevent side reactions, and minimize the harmful effects of toxic intermediates. A key advance in this field has been the production of empty MCP shells which were then filled with heterologous cargo by fusing targeting/ encapsulation sequences to desired enzymes/proteins (33,36,(60)(61)(62)(64)(65)(66)(67)(68). Further development of this approach for efficient encapsulation of multiple enzymes with defined stoichiometries will likely require the use of various types of targeting sequences, as well as parameterization of the molecular interactions that define encapsulation.…”

Section: Discussionmentioning

confidence: 99%

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

2017

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Bacterial microcompartments (MCPs) are extremely large proteinaceous organelles that consist of an enzymatic core encapsulated within a complex protein shell. A key question in MCP biology is the nature of the interactions that guide the assembly of thousands of protein subunits into a well-ordered metabolic compartment. In this report, we show that the N-terminal 37 amino acids of the PduB protein have a critical role in binding the shell of the 1,2-propanediol utilization (Pdu) microcompartment to its enzymatic core. Several mutations were constructed that deleted short regions of the N terminus of PduB. Growth tests indicated that three of these deletions were impaired MCP assembly. Attempts to purify MCPs from these mutants, followed by gel electrophoresis and enzyme assays, indicated that the protein complexes isolated consisted of MCP shells depleted of core enzymes. Electron microscopy substantiated these findings by identifying apparently empty MCP shells but not intact MCPs. Analyses of 13 site-directed mutants indicated that the key region of the N terminus of PduB required for MCP assembly is a putative helix spanning residues 6 to 18. Considering the findings presented here together with prior work, we propose a new model for MCP assembly.IMPORTANCE Bacterial microcompartments consist of metabolic enzymes encapsulated within a protein shell and are widely used to optimize metabolic process. Here, we show that the N-terminal 37 amino acids of the PduB shell protein are essential for assembly of the 1,2-propanediol utilization microcompartment. The results indicate that it plays a key role in binding the outer shell to the enzymatic core. We propose that this interaction might be used to define the relative orientation of the shell with respect to the core. This finding is of fundamental importance to our understanding of microcompartment assembly and may have application to engineering microcompartments as nanobioreactors for chemical production.

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Section: Discussionmentioning

confidence: 99%

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

2017

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“…The archetype of a prokaryotic cell lacks lipid-membrane enclosed subcellular organelles with few exceptions like magnetosomes or annamoxosomes [1,2]. Proteinaceous organelles lacking lipids, however, are widespread in prokaryotes where they often serve metabolic functions (metabolosomes) [2][3][4][5][6][7]. The prototype of this kind of subcellular biocatalytic entities is the carboxysome of cyanobacteria.…”

Section: Introductionmentioning

confidence: 99%

Engineering bacterial microcompartments with heterologous enzyme cargos

Wagner

Capitain

Richter

et al. 2016

Engineering in Life Sciences

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Bacterial microcompartments (BMCs) are intracellular proteinaceous organelles devoid of a lipid membrane that encapsulates enzymes of metabolic pathways. Salmonella enterica synthesizes propanediol‐utilization BMCs containing enzymes involved in the degradation of 1,2‐propanediol. BMCs can be designed to enclose heterologous proteins, paving the way to engineered catalytic microreactors. Here, we investigate broader applicability of this design principle by directing three different enzymes to the BMC. We demonstrate that β‐galactosidase, esterase Est5, and cofactor‐dependent glycerol dehydrogenase can be directed to the BMC and copurified with the microcompartment shell in a catalytically active form. We show that the BMC shell protects enzymes from pH‐dependent but not from temperature stress. Moreover, we provide evidence that the heterologously expressed BMCs act as a moderately selective diffusion barrier for lipophilic small molecules.

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“…(23)(24)(25)(26), as well as to heart disease and cancer in humans due to their metabolic roles in the gut microbiome (27)(28)(29)(30). Moreover, several labs have begun to develop MCPs as a platform for protein-based containers for use in renewable chemical production, drug delivery, and the expression of toxic proteins (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). However, as of yet, only three MCP types have been studied in any detail: the 1,2-propanediol utilization (Pdu) MCP, the ethanolamine utilization (Eut) MCP, and the carboxysome.…”

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confidence: 99%

A Bacterial Microcompartment Is Used for Choline Fermentation by Escherichia coli 536

et al. 2018

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Bacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization by and species. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogen 536. Growth studies indicated 536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization () gene cluster of 536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that the gene encodes a positive transcriptional regulator required for induction of the main operon in response to choline supplementation. Each of the 16 genes in the cluster was individually deleted, and phenotypes were examined. The ,, ,, ,, , and genes were required for choline degradation, but the remaining genes of the cluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed. Here, we investigate choline degradation in 536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation in 536, an organism for which sophisticated genetic analysis methods are available. In addition, the gene cluster of 536 is located in pathogenicity island II (PAI-II) and hence might contribute to pathogenesis.

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Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli

Cited by 52 publications

References 82 publications

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

Engineering bacterial microcompartments with heterologous enzyme cargos

A Bacterial Microcompartment Is Used for Choline Fermentation by Escherichia coli 536

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