Periodic Reporting for period 2 - SynBio4Flav (Synthetic microbial consortia-based platform for flavonoids production using synthetic biology) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project The one aim of this Project is the setup of a standardized pipeline for manufacturing flavonoids by using cutting edge synthetic biology tools and microbial chassis, supported by systems biology, synthetic biochemistry, protein engineering, and chemical engineering. SynBio4Flav endeavours not just to reassemble the biochemical pathways thereof but also to break down specific portions of the necessary biochemical routes and split them between different microbial species, each of them genetically programmed to deliver the optimal output for subsequent biosynthetic steps, i.e. distributed catalysis within an engineered microbial consortium.Ongoing efforts in SynBio are mainly targeted to the standardization of low complexity parts such as genes, operons and pathways while less attention has been paid to cellular systems and microbial communities. The current low yields obtained in the production of flavonoids calls for further progress in the optimization and standardization of the hierarchy abstraction of SynBio large-scale production, particularly at the high-complexity levels such as cell systems and microbial communities.Synbio4Flav focuses on the entire SynBio hierarchy abstraction chain, including i) DNA/proteins, enzymatic reactions and metabolic pathways (Objective 1), ii) cells (Objective 2), iii) microbial consortia (Objective 3) and iv) downstream processing (Objective 4). These four objectives match the scope of work packages 2, 3, 4 & 5, respectively. Despite being addressed individually, their inputs and outputs are narrowly intertwined within the overarching conceptual and technical framework of synthetic biology.In addition, two accessory WPs (WP6 and WP7) support optimization by providing the proper standardized and complementary SynBio tools and systems design & analysis required for optimal integration, respectively. Besides fuelling SynBio4Flav's optimization chain, WP6 and WP7 provide new SynBio and computational tools with potential applicability beyond this project, and thus have a strong merit on their own. In each of these WPs, several partners are contributing a synergistic set of tools and expertise to develop specific technologies needed for the implementation of the final envisioned synthetic microbial consortia-based platform for flavonoid production. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During this 2RP the synthetic pathways responsible for flavonoids biosynthesis and decoration optimized during the 1RP within WP2, have been largely implemented in optimized microbial chassis (WP3). The optimization of precursor, main core synthons and a large set of functionalized flavonoids have been already achieved. Novel computational and synthetic biology tools have been implemented and they are now being utilized to optimize the synthetic pathways within the target hosts providing dynamic control and system analysis of these microbial biocatalyst. The designing of the proper containers for the final synbio4flav consortia have been addressed through the development of semipermeable ceramic membranes which will be critical to design the two chambers bioreactors in WP4. Initial SOPs for fermentation and product purification have been establish providing a path for the feasible production of flavonoids using biotechnological approaches. Finally, a huge effort on dissemination and exploitation has already been done, thus placing the project in the showcase of microbial biotechnology and circular economy. 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 new approach proposed by SynBio4Flav includes the de-composition of flavonoid pathways into minimal modules and further re-assembly at microbial community level. In these first 36 months, the project has made significant progress towards this objective. Work done in WP2 and WP3 has demonstrated the suitability of optimizing precursor, assembly and decoration pathways in the context of different bacterial chassis, thus delivering a proof of concept of the SynBio4Flav approach. Furthermore, the development of new devices and mathematical models for the optimization of synthetic consortia developed in WP4 and WP7 pave the way for the validation of this new approach in the next reporting periods. Promising advances in the synthesis of basic flavonoid synthons have been achieved during this 2RP. In addition, the identification of new decoration enzymes such as new glucosidases and hydrolases will facilitate the achievement of SynBio4Flav's expected impacts, e.g. “production of at least 10 natural complex glycosylated flavonoids difficult to synthetize by chemical approaches and, at least, 5 novel glycosylated flavonoids”. SynBio4Flav has largely contributed to standardization in synthetic biology. So far, extension of SEVA standards toward genome editing in P. putida and S. albus and the design and validation of new standardized biological parts in the context of Modular Cloning have been done. Therefore, new promoters, enzymes and regulators have been standardized and will be publicly accessible through DOULIX www.doulix.comSynBio4Flav consortium has implemented a large array of dissemination actions contributing not only to put the project on the map, but also to bring synthetic biology and the benefits of flavonoids to society. We have been approached by a broad variety of stakeholders ranging from press reporters to school children (WP8). During this 2RP new and innovative actions facing the shortcoming imposed by COVID 19 pandemic have been implemented including and online exhibition and a podcast series highlighting the different aspect of the project and scientific and industrial partners. Society's overall reaction to the project has been very positive, which seems to confirm that our outreach efforts have also contributed to a public acceptance of synthetic biology and biotechnology as enabling technologies for solving societal challenges. The first scientific outputs are also being generated in the form of conference presentations and peer-reviewed publications. From plant- to biotech: Expected shift provided by Synbio4Flav in flavonoids production.