Dynamic and Single Cell Characterization of a CRISPR-Interference Toolset in Pseudomonas putida KT2440 for ..beta..-Ketoadipate Production from p-Coumarate

Jacob Fenster, Allison Werner, Jian Tay, Matthew Gillen, Leo Schirokauer, Nicholas Hill, Audrey Watson, Kelsey Ramirez, Christopher Johnson, Gregg Beckham, Jeffrey Cameron, Carrie Eckert

Research output: Contribution to journalArticlepeer-review

4 Scopus Citations

Abstract

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

Original languageAmerican English
Article numberArticle No. e00204
Number of pages11
JournalMetabolic Engineering Communications
Volume15
DOIs
StatePublished - Dec 2022

Bibliographical note

Publisher Copyright:
© 2022

NREL Publication Number

  • NREL/JA-2800-83098

Keywords

  • CRISPR interference
  • Heterogeneity
  • Microbial lignin valorization
  • Pseudomonas putida KT2440
  • Single-cell analysis

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