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 language | English |
|---|---|
| Article number | e00204 |
| Journal | Metabolic Engineering Communications |
| Volume | 15 |
| DOIs | |
| State | Published - Dec 2022 |
Funding
This manuscript has been authored in part by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).The genetic tractability of Pseudomonas putida KT2440 (hereafter P. putida), as well as its broad carbon metabolism and tolerance to diverse environmental and oxidative stressors, position this host as a promising chassis for biotechnological applications (Chavarría et al., 2013; Dos Santos et al., 2004; Nelson et al., 2002; Timmis, 2002). Specifically, interest in using P. putida to convert lignin-related aromatic compounds to value-added products has grown due to the ability of this host to catabolize diverse aromatic substrates and tolerate cytotoxic compounds in lignin depolymerization streams (Abdelaziz et al., 2016; Becker and Wittmann, 2019; Beckham et al., 2016; Linger et al., 2014; Salvachúa et al., 2015; Weiland et al., 2022). In addition to its robustness in lignin rich environments, P. putida has a wealth of genetic tools that enable researchers to easily delete or introduce genes for the purpose of functional genomics and strain engineering, which was recently reviewed (Martin-Pascual et al., 2021). Natural products such as polyhydroxyalkanoates (Linger et al., 2014; Liu et al., 2017; Salvachúa et al., 2020), muconic acid (Johnson et al., 2019; Kohlstedt et al., 2018; Salvachua et al., 2018; Sonoki et al., 2018), lactate (Johnson and Beckham, 2015), pyruvate (Johnson and Beckham, 2015), β-ketoadipate (βKA) (Johnson et al., 2019; Okamura-Abe et al., 2016), 2-pyrone-4,6-dicarboxyilc acid (Lee et al., 2022), among others (Incha et al., 2020; Johnson et al., 2019; Okamura-Abe et al., 2016) have been produced in defined media from model lignin aromatics as well as from crude lignin liquor through traditional knock in-knock out methods. The general metabolic engineering strategy used in these examples in which the molecular targets are intermediates in catabolic pathways relies upon generating mutants with partial pathways that terminate at a desired product. Alternate carbon sources such as glucose or acetate are supplemented into the media to support growth and cell maintenance given that the majority of carbon is retained in the bioproduct. As an example, Johnson et al. 2019 produced 41.1 g/L βKA at near theoretical yield (1 mol/mol) from 4-hydroxybenzoate (4-HBA), although this P. putida ΔpcaIJ production strain required co-feeding glucose to support growth.This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. CAE, JCC, JAF, AZW and GTB acknowledge funding from The Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Funding was provided to KJR, CWJ, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. This study was financially supported in part by the U.S. Department of Energy (DOE) DE-SC0019306 and DE-SC0020361to J.C.C. We thank Emily F. Freed for her critical reading of the manuscript. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. CAE , JCC , JAF , AZW and GTB acknowledge funding from The Center for Bioenergy Innovation , a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . Funding was provided to KJR, CWJ, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. This study was financially supported in part by the U.S. Department of Energy (DOE) DE-SC0019306 and DE-SC0020361to J.C.C.
| Funders | Funder number |
|---|---|
| DOE Public Access Plan | KT2440 |
| Johnson and Beckham | |
| U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office | DE-SC0019306 |
| U.S. Department of Energy Research Center | |
| U.S. Government | |
| U.S. Department of Energy | DE-AC36-08GO28308 |
| Office of Science | |
| Biological and Environmental Research | |
| National Renewable Energy Laboratory | |
| Center for Bioenergy Innovation | |
| βKA | 4-HBA |
Keywords
- CRISPR interference
- Heterogeneity
- Microbial lignin valorization
- Pseudomonas putida KT2440
- Single-cell analysis