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Synthetic biology-guided engineering of Pseudomonas putida for biofluorination

Periodic Reporting for period 2 - SinFonia (Synthetic biology-guided engineering of Pseudomonas putida for biofluorination)

Reporting period: 2020-07-01 to 2022-02-28

SinFonia aims to produce novel fluorochemicals in a biological way. Materials containing the element fluorine (F) are extremely important in our modern world, with applications in electronics, healthcare, automotive and wearables. Currently these fluorochemicals are exclusively synthesized using chemical methods, something SinFonia wants to change. The ambition of SinFonia is to set the stage for a future economically, ecologically and societally sustainable value chain for the production of novel, bio-based fluoropolymers from renewable substrates. SinFonia is designed as a truly interdisciplinary project. Project partners contribute with their specific knowledge and know-how to meet the needs of all stakeholders. There are 7 work packages in total, with 13 partners contributing to them. Each work package has been mapped out extensively for 4 years and includes detailed deliverables and milestones.
In order to build robust cell factories for the production of fluoropolymers, the Consortium is working on multiple, complementary aspects related to metabolic engineering and synthetic biology of bacterial cell factories towards establishing in vivo biofluorination.

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. Importantly, by using these tools developed for SinFonia, the consortium was able to isolate the fastest fluorinase known in Nature (described in detail in WP2, see preprint at https://www.biorxiv.org/content/10.1101/2022.03.08.483453v1.abstract).

Partners have also developed novel synthetic biology tools to facilitate genome engineering of Pseudomonas putida KT2440 towards whole-cell 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 have extensively re-wired 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; and the these devices have been successfully used to couple the activity of synthetic biofluorination modules to bacterial growth to drive evolution-guided optimization of the pathway.

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. Interestingly, there was no damage/instability identified in our engineered cell factories, which supports the validity of our general approach to establish biofluorination in living cells.

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 and the exploitation strategy is paving the way to Technology Readiness Level - TRL 6.
Communication activities have been carried out via multiple channels, adding new information such as a set of infographics (see: https://www.sinfoniabiotec.eu/project-infographics/) to our website and social media presence, we organized a series of citizen engagement and public perception events in five European countries, produced an interactive museum exhibition about biofluorination that is now public in the Science Museum in The Haague (NL) (see: https://www.sinfoniabiotec.eu/2022/01/12/traces-of-fluorine/) and completed the residency of artist and composer Eduardo Miranda in Pablo Nikel’s lab in Copenhagen, who produced a musical sinfonia based on biofluorination enzymes (see: https://www.sinfoniabiotec.eu/2021/06/22/music-from-dna-creating-a-sinfonia-inside-a-bacterial-cell/ and https://www.soundclick.com/music/songInfo.cfm?songID=14247892 ) .
The ambition of SinFonia is to set the stage for a future economically, ecologically and societally sustainable value chain for the production of novel, bio-based fluoropolymers from renewable substrates. The innovation potential of SinFonia is high: we have set several ground-breaking objectives that include cutting-edge synthetic biology, smart metabolic engineering, rational protein design, bioprocess engineering, material science and polymer chemistry. Our project will contribute concepts and approaches to these fields (including standardization of metabolic pathways from difficult-to-manipulate organisms to ease their transfer into a formatted and robust bacterial chassis) while designing cell factories for efficient synthesis of novel fluorinated products.
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