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

Julie E Chaves; Rosemarie Wilton; Yuqian Gao; Nathalie Munoz Munoz; Meagan C Burnet; Zachary Schmitz; John Rowan; Leah H Burdick; Joshua Elmore; Adam Guss; Dan Close; Jon K Magnuson; Kristin E Burnum-Johnson; Joshua K Michener

doi:10.1016/j.mec.2020.e00139

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

Metab Eng Commun. 2020 Jul 19:11:e00139. doi: 10.1016/j.mec.2020.e00139. eCollection 2020 Dec.

Authors

Affiliations

¹ Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008, MS6342 Oak Ridge, Tennessee, 37831-6342, USA.
² Argonne National Laboratory, USA.
³ Pacific Northwest National Laboratory, USA.

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.