Detailed description

SynBio4Flav develops a standardized pipeline for the surrogate production of plant flavonoids using synthetic microbial consortia (SMCs) for systems-guided assembly of such complex natural products. By facilitating component troubleshooting and reusability, instead of optimizing a single whole-cell biocatalyst, SynBio4Flav recreates the non-homogeneous scenario of natural flavonoid production processes. Specifically, it breaks down complex, highly regulated metabolic pathways into stand-alone modules and then assigns each module to a microorganism that is genetically engineered to deliver the optimal output for subsequent biosynthetic steps within the SMC.

SynBio4Flav’s groundbreaking approach impacts the whole synthetic biology hierarchy abstraction, i.e. from cell systems to microbial communities, and is expected to replace the more traditional use of single-cell biocatalysts.

Libraries of cell systems engineered to deliver optimal outputs and novel synthetic biology tools for using these cell systems to assemble 3D synthetic microbial consortia are among the envisaged project outputs. On this basis, SynBio4Flav develops a standardized platform containing hundreds of optimal cell systems for exploring the full combinatorial space of flavonoid biosynthesis, including thousands of new-to-nature analogues. SynBio4Flav is thus expected to reach a Technology Readiness Level (TRL) 5 in the production of natural and new-to-nature glycosylated flavonoids.

WP 1 – Project management and coordination

WP 1 provides overall management of the project to ensure it meets all contractual obligations timely and to a high standard. A strong focus is placed on meaningful communication within and outwith the consortium, an efficient liaison with the EC, open-access publications and the delivery of gender equality goals.

WP 2 – 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 also microbial enzymes and different regulatory elements and then assembled into new-to-nature biosynthetic pathways, finally leading to a higher flavonoid production.

Complex plants flavonoids biosynthetic pathways will be de-convoluted in minimal DNA parts and further re-assembled into synthetic functional units covering the whole biosynthetic pathways.

WP 3 – 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.

WP 4 – 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 of the selected compounds. Downstream processing of target compounds will be optimized to generate draft specification for application development, including the assessment of their potential benefits and risks.

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

WP 9 deals with ethics requirements attached to the research as set out in the Grant Agreement.