Production of a second phase of hydrophobic aromatics with solvent-tolerant Pseudomonas

Replacement of fossil chemicals with biological counterparts has been widely accepted as a vital pursuit to increase the sustainability of our chemical and material industries. Synthetic biology and metabolic engineering enable us to produce a plethora of chemicals with microbes, but the majority of these never make it past the proof-of-principle stage. This is especially the case for drop-in bulk aromatics like styrene or benzene. The main reason for this is that such products are too toxic to ordinary production microbes. In PROSPER I aim to overcome this hurdle and demonstrate the efficient microbial production of hydrophobic aromatic chemicals using solvent-tolerant Pseudomonas. I will engineer this unique extremophile to break the solubility barrier of these chemicals, forming a second phase of product. This second phase provides a virtually endless product sink and it enables extremely simple downstream recovery.The bio-based production of a second phase of such chemicals has thus far never been shown. I believe that this relates to a fundamental problem in biotechnology: production tolerance, i.e., tolerance of the producing organism to the produced product, rather than to an externally added chemical (as it is usually studied). In PROSPER I intend to generate deep mechanistic insights into the processes governing both types of tolerance and to leverage these insights to open up a new field of biotechnological production of hydrophobic compounds. To achieve this, I will develop new methods to analyze intracellular solvent concentrations, build a Pseudomonas chassis with enhanced production tolerance to hydrophobic solvents, and enable production of solvents like styrene, ethylbenzene, and even benzene.I am in a unique position to achieve this goal, with over 15 years of experience in the engineering of Pseudomonas as a workhorse in biotechnology, the study of solvent-tolerance, and the development and application of synthetic biology tools and metho