Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

• Published in: Metabolic engineering communications Vol. 11; no. C; p. e00139
• Main Authors: Chaves, Julie E., Wilton, Rosemarie, Gao, Yuqian, Munoz, Nathalie Munoz, Burnet, Meagan C., Schmitz, Zachary, Rowan, John, Burdick, Leah H., Elmore, Joshua, Guss, Adam, Close, Dan, Magnuson, Jon K., Burnum-Johnson, Kristin E., Michener, Joshua K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2020; Elsevier
• Subjects: BASIC BIOLOGICAL SCIENCES; fluorescence; fluorescent proteins; Full Length; gene expression; genes; genomics; loci; metabolic engineering; phenotype; protein synthesis; proteomics; Pseudomonas putida; synthetic biology; viability
• ISSN: 2214-0301; 2214-0301
• DOI: 10.1016/j.mec.2020.e00139

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. •Pseudomonas putida KT2440 chromosomal loci were characterized as potential insertion targets for heterologous genes.•Integration location had a significant effect on heterologous protein expression and host phenotype.•The identification of an appropriate chromosomal insertion location is essential to optimize genetic engineering design.