Abstract
The development of Pseudomonas strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in Pseudomonas putida KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.
| Original language | English |
|---|---|
| Article number | e00139 |
| Journal | Metabolic Engineering Communications |
| Volume | 11 |
| DOIs | |
| State | Published - Dec 2020 |
Funding
Research was sponsored by the US Department of Energy BioEnergy Technologies Office through the Agile Biofoundry. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. A portion of this research was performed at Pacific Northwest National Laboratory (PNNL) using EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. PNNL is a multiprogram national laboratory operated by Battelle for the Department of Energy (DOE) under Contract DE-AC05-76RLO 1830. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Research was sponsored by the US Department of Energy BioEnergy Technologies Office through the Agile Biofoundry. Oak Ridge National Laboratory is managed by UT-Battelle, LLC , for the U.S. DOE under contract DE-AC05-00OR22725 . A portion of this research was performed at Pacific Northwest National Laboratory (PNNL) using EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. PNNL is a multiprogram national laboratory operated by Battelle for the Department of Energy (DOE) under Contract DE-AC05-76RLO 1830 . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.