Periodic Reporting for period 3 - SynBio4Flav (Synthetic microbial consortia-based platform for flavonoids production using synthetic biology) Reporting period: 2022-01-01 to 2023-08-31 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.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 (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.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 SynBio4Flav, post-completion, pioneered distributed catalysis, enabling intricate chemical synthesis via synthetic microbial consortia. Notably, it successfully produced diverse, challenging-to-obtain functionalized flavonoids at a gram-scale. The project optimized fermentation using novel bioreactors and refined downstream processes, validating flavonoids’ health benefits through animal models. Additionally, it contributed significantly to synthetic biology standardization and advanced computer-assisted design for complex chemical processes.Objective 1 involved synthetic pathway optimization: A large array of enzymes involved in precursor biosynthesis, assembly and decoration have been identified and/or optimized using protein engineering and high-throughput screening drive by robotic platforms. Enzymes crucial for precursor biosynthesis, assembly, and decoration were enhanced, facilitating the production of coumarate and other flavonoid derivatives. Novel glycosyltransferases from fungi and gut bacteria and C-8 hydroxylation platforms enabling more lipophilic products were developed.Objective 2 focused on cell systems optimization: Several microbial strains were successfully engineered for the (over-) production of key flavonoid precursors, such as aromatic amino acids, phenylpropanoids, and malonate, at a gram-scale. A library of E. coli strains producing high levels of activated sugars suitable for flavonoid glycosylation was engineered and validated. Flavonoids structural diversification in S. albus has generated more than 35 different compounds. Objective 3 emphasized SMC optimization: Positive interactions between different species drove higher malonate titres stabilising the proof-of-concept of SMC as efficient biocatalysts. Naringenin, along with several glycosylated and hydroxylated derivatives, was de-novo synthesised using SMC. Yeast, S. cerevisiae, was engineered for the production of flavonoids as part of a microbial community. SMC fermentation was optimised from lab scale to industrial pilot scale.Objective 4 centered on downstream process optimization: Innovative bioreactors were built, and efficient transformation/decoration processes for flavonoids were achieved. Cataloging phytochemical standards expanded, and greener, cost-effective processes were implemented. They also evaluated flavonoid metabolism by human gut microbiota and conducted bioactivity tests showing strong antitumour and anti-inflammatory activities for some flavonoidsThe project also significantly contributed to standardization in synthetic and system biology, expanding tools like SEVA collections, DOULIX, and Modular Cloning Kit for synthetic biology. Additionally, it developed a computer-aided design platform for flavonoid biosynthesis, utilized artificial intelligence for enzyme activity prediction, and continuously improved genome-scale models for synthetic microbial consortia.As a result, more than 80 scientific papers were published during project duration. Animations on the project objectives, on flavonoids and synthetic biology were created. A virtual exhibition was installed on the project website in response to COVID19 restrictions. Three public conversations were held on the potential of metabolic engineering for providing solutions to global challenges. The podcast series ‘Made by Microbes’ was implemented to communicate key topics of the project. 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. 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. Furthermore, the development of new devices, mathematical models and novel biorreactor have allow the optimization of synthetic consortia developed in WP4 while work done on WP5 has validated Synbio4Flav approach at pre-industrial scale. Following this successful implementation of Synbio4flav expected impact e.g. “production of at least 10 natural complex glycosylated flavonoids difficult to synthetize by chemical approaches and, at least, 5 novel glycosylated flavonoids” has been achieved. SynBio4Flav has also 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. SynBio4Flav 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 the project 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.
