Flavonoids are compounds found in nature, synthesized through complex pathways involving intense chemical trafficking through plant compartments.

SynBio4Flav will breakdown specific portions of these complex pathways and distribute the highly regulated biochemical routes between different microbial species genetically programmed to deliver an optimal output.

SynBio4Flav pursues the implementation of a standardized pipeline for the surrogate production of plant flavonoids in synthetic microbial consortia (SMCs) by means of standardization, and systems-guided assembly of highly complex biological devices. By facilitating component troubleshooting and reusability, instead of optimizing a single whole-cell biocatalyst, SynBio4Flav will recreate the non-homogeneous scenario of natural flavonoid production by breaking-down specific portions of the complex pathways, and distributing the highly regulated biochemical routes between different microbial species genetically programmed to deliver an optimal output from the corresponding biosynthetic step(s).
Through enabling this novel approach, SynBio4Flav will push the existing boundaries of synthetic biology by acting along the whole synthetic biology hierarchy abstraction (from cell systems to microbial communities). By creating libraries of optimized cell systems programmed to deliver optimal outputs, and novel synthetic biology tools for assembling cell systems into 3-D synthetic microbial consortia, SynBio4Flav will reach a TRL5 in production of natural and new-to-nature glycosylated flavonoids.
SynBio4Flav will create a standardized platform containing hundreds of optimal cell systems for exploring the full combinatorial space of flavonoids biosynthesis, including thousands of new-to-nature analogues.

WP 1 – Project management and coordination
WP1 encompasses financial, administrative and contractual components, with specific focus on EU liaison, open-access publication, information exchange, organization of project meetings, supervision of implementation and progress, and gender equality.

WP2 – Design, assembly, and optimization of synthetic pathways for flavonoid production
Flavonoid production in plants can be viewed as a multi-step process including i) production of precursor molecules, ii) synthesis of the flavonoid and iii) functionalization/glycosylation of the flavonoid. In WP2, these steps are optimized first separately by using microbial enzymes and different regulatory elements and then assembled into new-to-nature biosynthetic pathways, finally leading to a higher flavonoid production. Additionally, enzymes for flavonoid diversification and transporters for optimized flavonoid ex- and import are studied and exploited to establish building blocks for efficient and diverse flavonoid production.
Genetic parts of flavonoid-producing plants are identified, optimized, and assembled into new-to-nature biosynthetic pathways. Genetic parts are to include enzymes for precursors, assembling/diversification and functionalization, regulators, and transporters, which will be used as bioreporters and chemicals trafficking optimization, respectively.

WP3 – Microbial chassis systems – Optimization and consolidation metabolism refactoring
Optimal performances of synthetic operons require specific systems design, including its contextualization within the cell host metabolism. The main goal of this WP is the cell chassis improvement towards the optimization of minimal flavonoid biosynthetic modules through large genome editing approaches. Bacterial chassis will be engineered optimizing resource allocation towards overproduction of key internal precursor metabolites. Subsequently, platform strains will be engineered for optimization of the production of central flavonoid synthons as well as the production of diversification flavonoids. Finally, the consolidation of individual cell systems will be achieved by expressing optimal precursor, assembly, diversification and functionalization modules within the context of these optimized bacterial chassis.
The performance of these minimal functional units will be systematically expressed and optimized using engineered microbial cell factories.

WP4 – Synthetic microbial consortia design, construction and optimization chemicals trafficking optimization
System design and validation of complex SMCs allowing the re-assembling of the minimal flavonoid modules designed and optimized in WP2 and WP3. For this complex model-based design, HTS evaluation and the exploration of novel spatial organisation of synthetic communities is addressed.
Flavonoids will be synthetized, a la carte, by the rational combination of these cell factories within the context of synthetic microbial communities.

WP5 – Validation & demonstration of microbial synthetic platform bottlenecks removal, fermentation optimization
Performance screening at lab scale of promising SMCs and validated feasibility of their potential production at the industrial pilot scale. Substantial knowledge is gained for the application development the selected compounds. A new SMC-based bioreactor will be designed, and fermentation protocols will be optimized to ensure consistent production of the selected products. Substrate consumption and product formation kinetics will be analysed and tuned by nutritional and operational approaches aiming at high-titer (for improved downstream processes) and yield (for reduced material costs). Downstream processing of target compounds will be optimized to generate draft specification for application development, including the assessment of their potential benefits and risks. Pharmacological, flavouring and functional effects on different evaluation platforms will be screened and comprehensively analysed.
The cost effectiveness of SynBio4Flav approach will be achieved by optimizing and further up-scaling the synthetic microbial consortia bioprocess at industrial scale.

WP6 – Standardization on synthetic biology
This WP will provide the required standardization along the hierarchy abstraction of SynBio including newly developed SynBioTools for efficient synthetic pathways and SMC assembling.

WP 7 – Systems biology and analysis
Genetic engineering for optimization of microbial chassis systems and synthetic microbial consortia in WP3 and WP4 will be through model-driven design, leveraging on the strength of system biology to handle large data. The functional states, characteristics and behaviours of the metabolic networks of all organisms will be evaluated and simulated through computational models of metabolism in this work package.

WP8 – Dissemination & exploitation
Scientific results of the SynBio4Flav project are communicated to relevant target audiences to foster scientific and public dialogue with the aim of identifying new applications.

WP9 – Ethics requirements
The objective is to ensure compliance with the ‘ethics requirements’ set out in this work package.