TY - JOUR
T1 - Dynamic and Single Cell Characterization of a CRISPR-Interference Toolset in Pseudomonas putida KT2440 for ..beta..-Ketoadipate Production from p-Coumarate
AU - Fenster, Jacob
AU - Werner, Allison
AU - Tay, Jian
AU - Gillen, Matthew
AU - Schirokauer, Leo
AU - Hill, Nicholas
AU - Watson, Audrey
AU - Ramirez, Kelsey
AU - Johnson, Christopher
AU - Beckham, Gregg
AU - Cameron, Jeffrey
AU - Eckert, Carrie
N1 - Publisher Copyright:
© 2022
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
KW - CRISPR interference
KW - Heterogeneity
KW - Microbial lignin valorization
KW - Pseudomonas putida KT2440
KW - Single-cell analysis
UR - http://www.scopus.com/inward/record.url?scp=85137117946&partnerID=8YFLogxK
U2 - 10.1016/j.mec.2022.e00204
DO - 10.1016/j.mec.2022.e00204
M3 - Article
AN - SCOPUS:85137117946
SN - 2214-0301
VL - 15
JO - Metabolic Engineering Communications
JF - Metabolic Engineering Communications
M1 - Article No. e00204
ER -