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Combinatorial pathway engineering using type I-E CRISPR interference

Biotechnol Bioeng. 2018 Jul;115(7):1878-1883. doi: 10.1002/bit.26589. Epub 2018 Mar 30.

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.

Keywords: 3-hydroxypropionic acid; CRISPR interference; combinatorial gene expression; malonyl-coA; metabolic flux engineering.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Bacterial
  • Genetic Variation
  • Lactic Acid / analogs & derivatives*
  • Lactic Acid / metabolism
  • Malonyl Coenzyme A / metabolism
  • Metabolic Engineering / methods*
  • Metabolic Networks and Pathways / genetics*
  • Recombination, Genetic

Substances

  • Lactic Acid
  • Malonyl Coenzyme A
  • hydracrylic acid