Streamlined Pseudomonas taiwanensis VLB120 Chassis Strains with Improved Bioprocess Features

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solvent-sensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis à la carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.