Cargo encapsulation in bacterial microcompartments: Methods and analysis

Taylor M. Nichols, Nolan W. Kennedy, Danielle Tullman-Ercek*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations

Abstract

Metabolic engineers seek to produce high-value products from inexpensive starting materials in a sustainable and cost-effective manner by using microbes as cellular factories. However, pathway development and optimization can be arduous tasks, complicated by pathway bottlenecks and toxicity. Pathway organization has emerged as a potential solution to these issues, and the use of protein- or DNA-based scaffolds has successfully increased the production of several industrially relevant compounds. These efforts demonstrate the usefulness of pathway colocalization and spatial organization for metabolic engineering applications. In particular, scaffolding within an enclosed, subcellular compartment shows great promise for pathway optimization, offering benefits such as increased local enzyme and substrate concentrations, sequestration of toxic or volatile intermediates, and alleviation of cofactor and resource competition with the host. Here, we describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as an enclosed scaffold for pathway sequestration and organization. We first describe methods for controlling Pdu MCP formation, expressing and encapsulating heterologous cargo, and tuning cargo loading levels. We further describe assays for analyzing Pdu MCPs and assessing encapsulation levels. These methods will enable the repurposing of MCPs as tunable nanobioreactors for heterologous pathway encapsulation.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc
Pages155-186
Number of pages32
DOIs
StatePublished - 2019

Publication series

NameMethods in Enzymology
Volume617
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Bacterial microcompartments (MCPs)
  • Encapsulation
  • Enzyme assays
  • Flow cytometry
  • Fluorescence microscopy
  • Metabolic engineering
  • Microcompartment purification
  • Protein scaffolds
  • Salmonella enterica serovar Typhimurium LT2
  • Targeting sequences

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Nichols, T. M., Kennedy, N. W., & Tullman-Ercek, D. (2019). Cargo encapsulation in bacterial microcompartments: Methods and analysis. In Methods in Enzymology (pp. 155-186). (Methods in Enzymology; Vol. 617). Academic Press Inc. https://doi.org/10.1016/bs.mie.2018.12.009