Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate
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 var...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-12-01
|
| Series: | Metabolic Engineering Communications |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S221403012200013X |
| _version_ | 1811178901055995904 |
|---|---|
| author | Jacob A. Fenster Allison Z. Werner Jian Wei Tay Matthew Gillen Leo Schirokauer Nicholas C. Hill Audrey Watson Kelsey J. Ramirez Christopher W. Johnson Gregg T. Beckham Jeffrey C. Cameron Carrie A. Eckert |
| author_facet | Jacob A. Fenster Allison Z. Werner Jian Wei Tay Matthew Gillen Leo Schirokauer Nicholas C. Hill Audrey Watson Kelsey J. Ramirez Christopher W. Johnson Gregg T. Beckham Jeffrey C. Cameron Carrie A. Eckert |
| author_sort | Jacob A. Fenster |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T06:26:50Z |
| format | Article |
| id | doaj.art-fb317168da06436b87446297bd132ca2 |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-04-11T06:26:50Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-fb317168da06436b87446297bd132ca22022-12-22T04:40:21ZengElsevierMetabolic Engineering Communications2214-03012022-12-0115e00204Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarateJacob A. Fenster0Allison Z. Werner1Jian Wei Tay2Matthew Gillen3Leo Schirokauer4Nicholas C. Hill5Audrey Watson6Kelsey J. Ramirez7Christopher W. Johnson8Gregg T. Beckham9Jeffrey C. Cameron10Carrie A. Eckert11Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, 80309, USA; Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USARenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USABioFrontiers Institute, 3415 Colorado Avenue, Boulder, CO, 80309, USARenewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USA; Department of Biochemistry, University of Colorado, Boulder, CO, 80309, USADepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138, USARenewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USA; Department of Biochemistry, University of Colorado, Boulder, CO, 80309, USARenewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USA; BioFrontiers Institute, 3415 Colorado Avenue, Boulder, CO, 80309, USARenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USARenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USARenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USARenewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USA; Department of Biochemistry, University of Colorado, Boulder, CO, 80309, USA; National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA; Corresponding author. Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO 80309, USA.Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Corresponding author. PO Box 2008, MS6060 Oak Ridge, TN 37831-6060.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.http://www.sciencedirect.com/science/article/pii/S221403012200013XCRISPR interferenceSingle-cell analysisMicrobial lignin valorizationPseudomonas putida KT2440Heterogeneity |
| spellingShingle | Jacob A. Fenster Allison Z. Werner Jian Wei Tay Matthew Gillen Leo Schirokauer Nicholas C. Hill Audrey Watson Kelsey J. Ramirez Christopher W. Johnson Gregg T. Beckham Jeffrey C. Cameron Carrie A. Eckert Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate Metabolic Engineering Communications CRISPR interference Single-cell analysis Microbial lignin valorization Pseudomonas putida KT2440 Heterogeneity |
| title | Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate |
| title_full | Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate |
| title_fullStr | Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate |
| title_full_unstemmed | Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate |
| title_short | Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate |
| title_sort | dynamic and single cell characterization of a crispr interference toolset in pseudomonas putida kt2440 for β ketoadipate production from p coumarate |
| topic | CRISPR interference Single-cell analysis Microbial lignin valorization Pseudomonas putida KT2440 Heterogeneity |
| url | http://www.sciencedirect.com/science/article/pii/S221403012200013X |
| work_keys_str_mv | AT jacobafenster dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT allisonzwerner dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT jianweitay dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT matthewgillen dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT leoschirokauer dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT nicholaschill dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT audreywatson dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT kelseyjramirez dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT christopherwjohnson dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT greggtbeckham dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT jeffreyccameron dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate AT carrieaeckert dynamicandsinglecellcharacterizationofacrisprinterferencetoolsetinpseudomonasputidakt2440forbketoadipateproductionfrompcoumarate |