Cargo encapsulation in bacterial microcompartments: Methods and analysis

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

Research output: Chapter in Book/Report/Conference proceedingChapter

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 - Jan 1 2019

Publication series

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

Fingerprint

Propylene Glycol
Encapsulation
Scaffolds
Organizations
Metabolic engineering
Metabolic Engineering
Poisons
Toxicity
Industrial plants
Assays
Tuning
Engineers
Costs and Cost Analysis
DNA
Substrates
Enzymes
Costs
Proteins

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

Cite this

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

Cargo encapsulation in bacterial microcompartments : Methods and analysis. / Nichols, Taylor M.; Kennedy, Nolan W.; Ercek, Danielle Tullman.

Methods in Enzymology. Academic Press Inc., 2019. p. 155-186 (Methods in Enzymology; Vol. 617).

Research output: Chapter in Book/Report/Conference proceedingChapter

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BT - Methods in Enzymology

PB - Academic Press Inc.

ER -

Nichols TM, Kennedy NW, Ercek DT. Cargo encapsulation in bacterial microcompartments: Methods and analysis. In Methods in Enzymology. Academic Press Inc. 2019. p. 155-186. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2018.12.009