A synthetic system for expression of components of a bacterial microcompartment

TY - JOUR

T1 - A synthetic system for expression of components of a bacterial microcompartment

AU - Sargent, Frank

AU - Davidson, Fordyce A.

AU - Kelly, Ciarán L.

AU - Binny, Rachelle

AU - Christodoulides, Natasha

AU - Gibson, David

AU - Johansson, Emelie

AU - Kozyrska, Katarzyna

AU - Licandro Lado, Lucia

AU - MacCallum, Jane

AU - Montague, Rachel

AU - Ortmann, Brian

AU - Owen, Richard

AU - Coulthurst, Sarah J.

AU - Dupuy, Lionel

AU - Prescott, Alan R.

AU - Palmer, Tracy

PY - 2013/11

Y1 - 2013/11

N2 - In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose; that is to concentrate specific enzymatic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this work, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilisation (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T, and -U, and each were shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins were also designed and tested. Engineered hexa-Histidine tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

AB - In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose; that is to concentrate specific enzymatic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this work, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilisation (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T, and -U, and each were shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins were also designed and tested. Engineered hexa-Histidine tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

U2 - 10.1099/mic.0.069922-0

DO - 10.1099/mic.0.069922-0

M3 - Article

C2 - 24014666

SN - 1350-0872

VL - 159

SP - 2427

EP - 2436

JO - Microbiology

JF - Microbiology

IS - 11

ER -