Abstract - Probanowski

We target the bio-based production of hydrophobic aromatics, such as styrene or ethylbenzene, addressing major challenges, like climate change and environmental pollution. However, the toxicity of these products requires highly solvent tolerant biotechnological chassis to ensure the efficient production of hydrophobic aromatics. Pseudomonas taiwanensis VLB120 is a well-suited host for this approach, due to its native solvent tolerance mechanisms, genetic tractability, and the availability of streamlined production chassis. The primary mechanism of solvent toxicity is the accumulation of hydrophobic molecules in the inner cell membrane, thereby inducing membrane destabilization. Pseudomonads tackle solvent stress mainly through the solvent extrusion from the cell into the environment by solvent efflux pumps. In order to gain a deeper mechanistic insight into the tolerance mechanisms to intracellularly produced rather than externally added chemicals, fluorescent biosensors have been developed and applied. These biosensors were constructed based on the transcriptional regulators of solvent efflux pumps genes, which are highly responsive to intracellular aromatic solvents. To normalize the biosensor readout to cell fitness under solvent stress, a second constitutively expressed fluorescent protein has been integrated into the construct. The efficacy of this ratiometric fluorescent biosensor in monitoring intracellular solvent concentrations, and the impact of the solvent efflux pump ttgGHI expression levels on these concentrations, have been demonstrated. It was shown that the expression of ttgGHI, in the context of increasing promoter strength, resulted in declining biosensor readouts, signifying a decrease in the intracellular solvent concentrations with increasing quantities of TtgGHI. Furthermore, it was demonstrated that Pseudomonas strains with lower intracellular solvent concentrations were associated with an enhanced tolerance to externally added solvents. The present study focuses on elucidating the cellular differences between externally added and intracellularly produced hydrophobic aromatics based on whole cell biosensors in co-culture experiments with the aim of identifying cellular factors specific for production tolerance.

Tobias Probanowski, Jakob Rönitz, Benedikt Wynands and Nick Wierckx (Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany)