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

Project Information

PROSPER

Grant agreement ID: 101044949

DOI

10.3030/101044949

EC signature date 2 May 2022

Start date 1 January 2023

End date 31 December 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 999 485,00

EU contribution

€ 1 999 485,00

Coordinated by

FORSCHUNGSZENTRUM JULICH GMBH
Germany

Periodic Reporting for period 1 - PROSPER (Production of a second phase of hydrophobic aromatics with solvent-tolerant Pseudomonas)

Reporting period: 2023-01-01 to 2025-06-30

Summary of the context and overall objectives of the project

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 and ethylbenzene.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The implementation of the SIGHT system is a breakthrough in that it enables us to greatly increase our experimental throughput. Previously, the state of the art in solvent-tolerance cultures were shake flask cultures with manual sampling performed through a gas-tight valve with a syringe. This was extremely labour-intensive and also dangers due to the use of needles, limiting experimental throughput for growth curves to a few dozen cultures with low sampling resolution at best. SIGHT enables us to record growth curves of hundreds of cultures at once without manual sampling, making it a major enabler in our research.
The newly developed biosensors also give unprecedented insight into especially hydrophobic compounds, which were thus far not well studied in literature. With these biosensors, we are now generating the first quantitative proof-of-principle for measuring the effect of efflux pump expression on intracellular stressor concentrations.