Targeted Gene Repression to Optimize 2-pyrone 4,6-dicarboxylic acid (PDC), β-ketadipate (βKA), and Muconate Titers in Pseudomonas putida KT 2440

University of Colorado Boulder Background
The programmability of CRISPR-Cas machinery enables library based high-throughput and multiplexed experiments that have allowed for rapid genotype-phenotype mapping (Peters et al., 2016), protein engineering (Garst et al., 2017), strain engineering (Tarasava, Liu, Garst, & Gill, 2018), and gene discovery (Shalem et al., 2014). Both CRISPR-Cas editing and interference have been demonstrated in the bacteria Pseudomonas putida KT2440, a promising candidate for the industrial production of renewable chemicals from lignin. Although these studies establish the foundation for CRISPR-tool development in P. putida, the need remains for characterization and optimization of these tools before they can be leveraged for high-throughput and multiplexed experiments
Technology Overview
Researchers at the University of Colorado have discovered a method to optimize CRISPR-interference (CRISPRi) by screening inducible promoter systems that express catalytically-dead spCas9. This system enables higher transformation efficiencies in bacteria that grow at a wide range of temperatures, allowing for faster genetic engineering of a wider array of microbes than current available technologies. The arabinose inducible promoter system performed best and was used to image the repression of the essential division protein ftsZ in real time. Future studies will quantify the dynamic range of repression by targeting a genomically integrated fluorescent reporter gene. In addition, this toolset will be used to increase 2-pyrone 4,6-dicarboxylic acid (PDC), β-ketadipate (βKA), and muconate titers by temporally repressing downstream enzymes that funnel these desired products into central metabolism. To optimize CRISPR-Cas gene editing, the transformation protocol from Sun et al. 2018 was improved to increase the colony forming units by 100-fold while keeping the editing efficiency at 100%. The minimum homology arm (HA) length requirements for gene deletion was determined and the HA length requirements for gene integration and introducing single codon mutations are underway.
Further Details:
CRISPR-Tool Development in Pseudomonas putida KT2440 for High-Titer Strain Engineering and Multiplexed Approaches
Stage of Development

Enables higher transformation efficiencies
Faster genetic engineering on a wider array of microbes


Gene editing
Genetic research
Genotype-phenotype mapping
Gene discovery
Strain/protein engineering
Production of renewable chemicals

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