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 language | English (US) |
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Title of host publication | Methods in Enzymology |
Publisher | Academic Press Inc |
Pages | 155-186 |
Number of pages | 32 |
DOIs | |
State | Published - 2019 |
Publication series
Name | Methods in Enzymology |
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Volume | 617 |
ISSN (Print) | 0076-6879 |
ISSN (Electronic) | 1557-7988 |
Funding
The authors would like to thank Lisa Burdette, Emily Hartman, and Svetlana Ikonomova for helpful comments during the preparation of this chapter. This work was supported by the Army Research Office (grant W911NF-16-1-0169 to D.T.E.), the Department of Energy (grant DE-SC0019337 to D.T.E.), and the National Science Foundation (RAISE grant CBET-1844336 to D.T.E.). T.M.N. and N.W.K. were supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1842165. N.W.K. was supported in part by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University's Biotechnology Training Program.
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