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 flavonoids
The 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.
