Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate
Abstract
We report 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 hour 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 themore »
- Authors:
-
[5];
- University of Colorado, Boulder, CO (United States)
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- BioFrontiers Institute, Boulder, CO (United States)
- Harvard University, Cambridge, MA (United States)
- University of Colorado, Boulder, CO (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office
- OSTI Identifier:
- 1885323
- Alternate Identifier(s):
- OSTI ID: 1886879
- Report Number(s):
- NREL/JA-2800-83098
Journal ID: ISSN 2214-0301
- Grant/Contract Number:
- AC05-00OR22725; AC36-08GO28308; SC0019306; SC0020361
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Metabolic Engineering Communications
- Additional Journal Information:
- Journal Volume: 15; Journal Issue: NA; Journal ID: ISSN 2214-0301
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; CRISPR interference; single-cell analysis; microbial lignin valorization; Pseudomonas putida; KT2440; heterogeneity; Pseudomonas putida KT2440
Citation Formats
Fenster, Jacob A., Werner, Allison Z., Tay, Jian Wei, Gillen, Matthew, Schirokauer, Leo, Hill, Nicholas C., Watson, Audrey, Ramirez, Kelsey J., Johnson, Christopher W., Beckham, Gregg T., Cameron, Jeffrey C., and Eckert, Carrie A. Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate. United States: N. p., 2022.
Web. doi:10.1016/j.mec.2022.e00204.
Fenster, Jacob A., Werner, Allison Z., Tay, Jian Wei, Gillen, Matthew, Schirokauer, Leo, Hill, Nicholas C., Watson, Audrey, Ramirez, Kelsey J., Johnson, Christopher W., Beckham, Gregg T., Cameron, Jeffrey C., & Eckert, Carrie A. Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate. United States. https://doi.org/10.1016/j.mec.2022.e00204
Fenster, Jacob A., Werner, Allison Z., Tay, Jian Wei, Gillen, Matthew, Schirokauer, Leo, Hill, Nicholas C., Watson, Audrey, Ramirez, Kelsey J., Johnson, Christopher W., Beckham, Gregg T., Cameron, Jeffrey C., and Eckert, Carrie A. Sun .
"Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate". United States. https://doi.org/10.1016/j.mec.2022.e00204. https://www.osti.gov/servlets/purl/1885323.
@article{osti_1885323,
title = {Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate},
author = {Fenster, Jacob A. and Werner, Allison Z. and Tay, Jian Wei and Gillen, Matthew and Schirokauer, Leo and Hill, Nicholas C. and Watson, Audrey and Ramirez, Kelsey J. and Johnson, Christopher W. and Beckham, Gregg T. and Cameron, Jeffrey C. and Eckert, Carrie A.},
abstractNote = {We report 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 hour 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.},
doi = {10.1016/j.mec.2022.e00204},
journal = {Metabolic Engineering Communications},
number = NA,
volume = 15,
place = {United States},
year = {Sun Aug 28 00:00:00 EDT 2022},
month = {Sun Aug 28 00:00:00 EDT 2022}
}
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