Periodic Reporting for period 1 - SinFonia (Synthetic biology-guided engineering of Pseudomonas putida for biofluorination)
Reporting period: 2019-01-01 to 2020-06-30
During the first year, partners have successfully developed two novel bioinformatic tools which have been used for the design and identification of novel enzymes in the synthetic biofluorination pathway. The two tools are (i) EnzymeMiner, a web server for the identification of novel enzymes (https://loschmidt.chemi.muni.cz/enzymeminer/) and (ii) FireProtASR, a web server for automized ancestral sequence reconstruction (https://loschmidt.chemi.muni.cz/fireprotasr/). Thanks to these tools for in silico analysis, we identified catalytically efficient enzymes or protein modifications expected to improve their performance. Synthetic modules containing biofluorination enzymes have been implanted in P. putida and successfully tested for fluorometabolite synthesis.
Partners have also developed novel synthetic biology tools to facilitate genome engineering of Pseudomonas putida KT2440 towards biocatalysis and metabolic engineering. A versatile, robust, and user-friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in just 3-5 days has been designed, formatted and standardized. By adopting this toolbox, we are currently extensively re-wiring the central carbon metabolism of P. putida to accommodate the synthetic biofluorination pathway. Furthermore, the possibility to perform long-term adaptive laboratory evolution of P. putida has been confirmed in cultivation devices; such studies will be performed in the next months.
At the same time, we have assessed potential metabolic damage brought about by biofluorination in P. putida. This step is important to improve tolerance and genetic stability of P. putida cell factories to fluorinated compounds and ensure the long-term performance under industrial operating conditions.
Finally, to gain a complete picture of the sustainability of fluoropolymers production from renewable substrates by bioengineered P. putida, we are now thoroughly assessing all the environmental, economic and societal aspects of the value-chains of SinFonia. All the partners are disseminating the project at scientific platforms and international events; communication activities have adopted multiple channels to reach different targets of society (https://www.youtube.com/watch?v=HSjS6mOzZIo) and the exploitation strategy is paving the way to Technology Readiness Level - TRL 6.