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Title: 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:
 [1];  [2];  [3]; ORCiD logo [4]
  1. Chemical and Biological Engineering Department University of Colorado Boulder Boulder Colorado
  2. Renewable and Sustainable Energy Institute (RASEI) University of Colorado Boulder Boulder Colorado
  3. Muse Biotechnology Boulder Colorado
  4. 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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1002/bit.26589

Citation Metrics:
Cited by: 13 works
Citation information provided by
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