Abstract - Redeker

To support the transition toward a sustainable bioeconomy, our research focuses on Pseudomonas taiwanensis VLB120, engineered for efficient biosynthesis of hydrophobic aromatic compounds within a second-phase system. Through the PROSPER project, we aim to develop a scalable, sustainable, bio-based production platform, providing a greener alternative to conventional chemical manufacturing. Due to toxic effects of hydrophobic compounds, we focus on enhancing solvent tolerance in the bacterium applying various strategies. One of the approaches is the reduction of the cell surface hydrophobicity (CSH) of the chassis strain, thereby mitigating negative cell-solvent interactions. In our work on chassis engineering, we developed a novel methodology that integrates bacterial cell distribution within a two-phase system with adaptive laboratory evolution (ALE). This approach enabled us to actively modulate the bacterial cell envelope and its membrane composition to achieve a desired hydrophobicity. Starting from a genome-reduced chassis strain, we successfully minimized cell surface hydrophobicity by 20 %. Genetic analysis revealed that mutations in a response regulator gene were responsible for this altered phenotype. Notably, transcriptome analysis demonstrated significant downregulation of genes associated with type VI secretion systems in Pseudomonas, providing valuable insights into the molecular mechanisms underlying the reduced hydrophobicity. The resulting mutant strains exhibited advantageous phenotypes, including decreased biofilm formation, lower emulsification capacity, and reduced membrane vesicle production. These findings validate ALE as a powerful tool for fine-tuning cell surface properties including the membrane composition. The altered membrane properties can be linked to improved bioprocess performance. Given their enhanced suitability for industrial applications— particularly in two-phase cultivations and downstream processing—these engineered strains represent a significant step toward more efficient, biotechnology-driven production of aromatic solvents.

Till Redeker, Benedikt Wynands and Nick Wierckx (Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany)