In the first reporting period, we have mainly focused on method development to provide a basis for studying and enhancing solvent production tolerance. With these methods, we already gained remarkable insights into the fundamental basis of solvent tolerance, and how it relates to the production of solvents, in contrast to the external addition of solvents. Specifically, we made considerable progress in the following areas:
1. Engineer novel synthetic metabolic pathways to produce important aromatic solvents.
We have successfully implemented synthetic production pathways for several hydrophobic aromatics, including, among others, styrene, hydroxystyrene, and ethylphenol. These pathways were functionally expressed in streamlined chassis strains of the solvent-tolerant Pseudomonas taiwanensis and production of the compounds was demonstrated.
2. Measure intracellular solvent concentrations using fluorescent biosensors
We have established fluorescent biosensors for the accurate quantitative analysis of intracellular solvent concentrations for a range of different compounds. With these sensors, we have quantified the effect of different strain backgrounds, with different levels of solvent-tolerance, on the intracellular solvent concentration. This enables us to pinpoint key determinants of solvent tolerance, and what governs intracellular solvent concentrations.
3. Enhance production tolerance in solvent-producing strains using laboratory evolution
We were successful in re-routing central carbon metabolism through aromatic production pathways. The resulting strains were evolved, and we are currently analyzing the resulting mutants.
4. Engineer a streamlined P. taiwanensis chassis featuring a higher solvent production tolerance
Building on previous streamlining work, we further enhanced the tolerance of P. taiwanensis to a 2nd phase of solvents through a combination of genome reduction and laboratory evolution. These strains also show great promise in improving the handling of microbes in biphasic cultivation.
5. Use energy-yielding co-substrates to support solvent-tolerance
First experiments were performed to provide additional energy to P. taiwanensis under stress by co-feeding formic acid. We also explored the possibility to use ethylene glycol as co-substrate with higher potential energy yield.
Beyond these specific objectives, we also developed and validated the SIGHT system: A System for Solvent-Tight Incubation and Growth Monitoring in High Throughput. This system encompasses cultivation of solvent-tolerant P. taiwanensis in gas-tight glass GC vials in a 3D-printed rack which is compatible with standard 24-deepwel plate dimension. This enabled automated online monitoring of growth in our Growth Profiler system in the presence of volatile solvent. This cultivation system greatly increases our experimental throughput, both for growth and tolerance characterization, as well as for solvent production experiments.
