Combinatorial pathway engineering using type I‐E CRISPR interference
Abstract
ABSTRACT Optimization of metabolic flux is a difficult and time‐consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial‐and‐error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I‐E CRISPR‐Cas system and an iterative cloning strategy for construction of guide RNA arrays. This approach allowed us to build a combinatorial gene expression library three orders of magnitude larger than previous studies. In less than 1 month, we generated ∼12,000 combinatorial gene expression variants that target six different genes and screened these variants for increased malonyl‐CoA flux and 3‐hydroxypropionate (3HP) production. We were able to identify a set of variants that exhibited a significant increase in malonyl‐CoA flux and up to a 98% increase in 3HP production. This approach provides a fast and easy‐to‐implement strategy for engineering metabolic pathway flux for development of industrially relevant strains, as well as investigation of fundamental biological questions.
- Authors:
-
- Chemical and Biological Engineering Department University of Colorado Boulder Boulder Colorado
- Renewable and Sustainable Energy Institute (RASEI) University of Colorado Boulder Boulder Colorado
- Muse Biotechnology Boulder Colorado
- Chemical and Biological Engineering Department University of Colorado Boulder Boulder Colorado, Renewable and Sustainable Energy Institute (RASEI) University of Colorado Boulder Boulder Colorado
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1430735
- Grant/Contract Number:
- DE‐SC0008812
- Resource Type:
- Publisher's Accepted Manuscript
- Journal Name:
- Biotechnology and Bioengineering
- Additional Journal Information:
- Journal Name: Biotechnology and Bioengineering Journal Volume: 115 Journal Issue: 7; Journal ID: ISSN 0006-3592
- Publisher:
- Wiley Blackwell (John Wiley & Sons)
- Country of Publication:
- United States
- Language:
- English
Citation Formats
Tarasava, Katia, Liu, Rongming, Garst, Andrew, and Gill, Ryan T. Combinatorial pathway engineering using type I‐E CRISPR interference. United States: N. p., 2018.
Web. doi:10.1002/bit.26589.
Tarasava, Katia, Liu, Rongming, Garst, Andrew, & Gill, Ryan T. Combinatorial pathway engineering using type I‐E CRISPR interference. United States. https://doi.org/10.1002/bit.26589
Tarasava, Katia, Liu, Rongming, Garst, Andrew, and Gill, Ryan T. Fri .
"Combinatorial pathway engineering using type I‐E CRISPR interference". United States. https://doi.org/10.1002/bit.26589.
@article{osti_1430735,
title = {Combinatorial pathway engineering using type I‐E CRISPR interference},
author = {Tarasava, Katia and Liu, Rongming and Garst, Andrew and Gill, Ryan T.},
abstractNote = {ABSTRACT Optimization of metabolic flux is a difficult and time‐consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial‐and‐error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I‐E CRISPR‐Cas system and an iterative cloning strategy for construction of guide RNA arrays. This approach allowed us to build a combinatorial gene expression library three orders of magnitude larger than previous studies. In less than 1 month, we generated ∼12,000 combinatorial gene expression variants that target six different genes and screened these variants for increased malonyl‐CoA flux and 3‐hydroxypropionate (3HP) production. We were able to identify a set of variants that exhibited a significant increase in malonyl‐CoA flux and up to a 98% increase in 3HP production. This approach provides a fast and easy‐to‐implement strategy for engineering metabolic pathway flux for development of industrially relevant strains, as well as investigation of fundamental biological questions.},
doi = {10.1002/bit.26589},
journal = {Biotechnology and Bioengineering},
number = 7,
volume = 115,
place = {United States},
year = {Fri Mar 30 00:00:00 EDT 2018},
month = {Fri Mar 30 00:00:00 EDT 2018}
}
https://doi.org/10.1002/bit.26589
Web of Science
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