Abstract
Subcellular organization is critical for isolating, concentrating, and protecting biological activities. Natural subcellular organization is often achieved using colocalization of proteins on scaffold molecules, thereby enhancing metabolic fluxes and enabling coregulation. Synthetic scaffolds extend these benefits to new biological processes and are typically constructed from proteins or nucleic acids. To expand the range of available building materials, we use a minimal set of components from the lipid-encapsulated bacteriophage ϕ6 to form synthetic lipid-containing scaffolds (SLSs) in E. coli. Analysis of diffusive behavior by particle tracking in live cells indicates that SLSs are >20 nm in diameter; furthermore, density measurements demonstrate that SLSs contain a mixture of lipids and proteins. The fluorescent proteins mCitrine and mCerulean can be colocalized to SLSs. To test for effects on enzymatic production, we localized two enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent increase in indigo production, showing that SLSs can enhance the production of a commercially relevant metabolite.
Highlights
Introduction: (~620/750 words, no sub-headings) Intracellular protein localization is an important mechanism for regulating the function and activity of a variety of cellular processes
Since engineered scaffolds made from proteins or nucleic acids have been effectively used to enhance the activity of various enzymes or signaling proteins to create novel cellular functions[6,7,8,9,10]
Both classes of scaffolds have limitations, : protein scaffolds are limited in spatial complexity, and RNA scaffolds are intrinsically susceptible to enzymatic degradation in the absence of chemical modification[11]
Summary
Introduction: (~620/750 words, no sub-headings) Intracellular protein localization is an important mechanism for regulating the function and activity of a variety of cellular processes. We report the production of a new class of scaffolds that contain both proteins and lipids. We design and build synthetic lipid-containing scaffolds (SLSs) in E. coli using parts derived from the bacteriophage φ6 (Fig. 1A).
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