Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis.
Sebastian Köbbing1, Thorsten Lechtenberg2, Benedikt Wynands2
1Aachen Biology and Biotechnology-ABBt, Institute of Applied Microbiology-iAMB, RWTH Aachen University, 52074 Aachen, Germany.
ACS Synthetic Biology
|July 5, 2024
Summary
Researchers identified new genomic landing pads for Pseudomonas putida, enabling stable, condition-independent gene expression. This expands the genetic toolbox for advanced metabolic engineering in Pseudomonads.
Area of Science:
- Microbiology
- Synthetic Biology
- Genomics
Background:
- Genomic integration is crucial for engineering microbial production hosts.
- Limited characterized integration sites hinder advanced strain engineering requiring multiple gene insertions.
Purpose of the Study:
- To identify and characterize novel genomic integration sites (landing pads) in *Pseudomonas putida* KT2440.
- To enable condition-independent heterologous gene expression for advanced metabolic engineering.
Main Methods:
- RNA-Seq data analysis to identify genomic regions with stable expression patterns.
- Homologous recombination to insert reporter gene cassettes into identified intergenic sites.
- Locus-dependent expression analysis using insulated probe sensors.
Main Results:
- Ten novel landing pads were identified and characterized in *P. putida* KT2440.
- High reproducibility of expression was observed within individual landing pads.
- Significant variations in expression levels were found between different landing pads, indicating genomic context influence.
- Four landing pads were validated in *Pseudomonas taiwanensis* VLB120.
Conclusions:
- Genomic context significantly impacts gene expression, creating 'hot' and 'cold' spots.
- The identified landing pads provide valuable tools for enhanced metabolic engineering in Pseudomonads.
- This work expands the available genomic sites for stable and predictable gene expression.
