Combinatorial pathway engineering using type I‐E CRISPR interference

Tarasava, Katia; Liu, Rongming; Garst, Andrew; Gill, Ryan T.

doi:10.1002/bit.26589

Title: Combinatorial pathway engineering using type I‐E CRISPR interference

Journal Article · Fri Mar 30 00:00:00 EDT 2018 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.26589· OSTI ID:1430735

Tarasava, Katia ^[1]; Liu, Rongming ^[2]; Garst, Andrew ^[3];

^[4]

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

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.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: DE‐SC0008812

OSTI ID:: 1430735

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Vol. 115 Journal Issue: 7; ISSN 0006-3592

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 13 works

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