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Synthetic Circuits for Robust Orthogonal Production

Periodic Reporting for period 2 - SynCrop (Synthetic Circuits for Robust Orthogonal Production)

Reporting period: 2020-01-01 to 2021-12-31

At present, the exploitation of the potential of synthetic biology is greatly hindered by our limited ability to predict how production modules, also called synthetic circuits, will behave when implemented in living cells. SynCrop consortium has assembled a multi-disciplinary and trans-sectoral team of internationally renowned academic and industrial partners to challenge this major obstacle of synthetic biology. SynCrop’s major scientific goal was to rewire the cellular regulation into controllable and modular circuitry for efficient production of food additives (exopolysaccharides, vitamins, and carotenoids). The research of SynCrop harnessed the advances in various high-resolution technologies augmented with innovative modelling at multiple scales to quantitatively address the largely unexplored behavior of host cell in conjunction with production circuit towards (re)-designing the host for high yield production. We generated a suite of production circuits for some of the most important industrial organisms used in food industry (Escherichia coli, Streptococcus thermophilus and Saccharomyces cerevisiae). By monitoring imbalances in cellular homeostasis and physiological integrity and assessing distribution of cellular resources between synthetic circuits and housekeeping activities, we identified key set points to control the burden of synthetic circuit expression. These readouts were critical for creating the framework for reprogramming cellular behavior towards increasing yield of the engineered synthetic circuits under industrial settings and for robust and reliable operation of the host cells under varying environments.

SynCrop results and achievements fill in an important void in the European economic mission by efficiently exploiting the biosynthesis routes of industrially valuable products (exopolysaccharides, vitamins, and carotenoids). These three industrial products have wide range of applications, including dairy, animal and human nutrition, applications in cosmetics and pharmaceutical industries, yet using tedious, unsustainable and environmentally unfriendly methods.

Furthermore, SynCrop educated a new cohort of 15 international young researchers with unique combination of skills in quantitative biology that can construct modular and tunable synthetic circuits and reprogram organisms towards application-driven goals – an essential skillset for the young generation to become future research leaders or entrepreneurs in the emerging field of synthetic biology.
SynCrop’s research was dedicated to the main scientific objective - to control core cellular regulation by creating synthetic genetic parts and circuitry that are modular and can be easily rewired to support highly efficient production of food additives (exopolysaccharides, vitamins, and carotenoids). Key results include:

- Production and production-sensing circuits to produce exopolysaccharides, vitamins, or carotenoids for some of the most important industrial organisms used in food industry (Escherichia coli, Streptococcus thermophilus and Saccharomyces cerevisiae) were designed. The design and integration of the production circuits were optimized using model-guided studies.
- Alterations in the physico-chemical properties of the cytosol and membrane integrity upon operation of synthetic circuits were assessed through integrated state-of-the-art experimental and modelling approach.
- The integrative response of transcription, translation and metabolic responses upon operation of synthetic circuits were determined using various cutting-edge omics approaches combined with predictive modeling with different granularity.
- Various sensors to quantify homeostatic alterations were designed. The output of the production-sensing circuits together with the assessed limitations of core cellular processes served to define key set points used to mitigate the effect of the synthetic circuits.
- Through the precise control of expression of circuit components to economically use cellular resources the productivity of the synthetic circuits that produce exopolysaccharides, vitamins or carotenoids was optimized and their productivity enhanced in both laboratory and industrial scales.

From launching on the consortium, SynCrop has been present through a Wordpress-based website (http://www.synsrop.org/syncrop/) representing SynCrop’s own digital identity serving as a public window to present the consortium program and research. SynCrop has been disseminating results mainly through two channels: (1) academic channel, and (2) general public channel. So far, the SynCrop scientists have been extraordinarily successful and productive in their research - 27 peer-reviewed publications have been published and two IPs have been filed. The scientific results were disseminated through openly accessible publications, including pre-prints deposition on bioRxiv that enables immediate dissemination of results even during the peer review process. Each ESR presented the results at multiple national and international scientific conferences and workshops, both in presence and during the pandemic in a virtual format. Through participation in multiple public events, the ESRs enlarged the outreach of their scientific activities to layman audience. Several activities and events in schools were organized to raise the awareness and popularity of natural and engineering sciences among pupils - the next generation of students and scientists.
The researches in the SynCrop consortium used novel cutting-edge approaches and developed new synthetic biological parts, constructed archetypal genetic circuits, and rewired key production organisms (E. coli, S. thermophilus and S. cerevisiae) to enable efficient industrial production of important industrial food additives (exopolysaccharides, vitamins, or carotenoids). The solutions (e.g. circuit design optimization and host-cell rewiring to maximize production) are adoptable and applicable to other industrial products that are produced with single cellular hosts and thus, could impactfully enhance production schemes.

The ESRs used cutting-edge methodologies and gained expertise in the systems and production behavior of the most important industrial organisms towards innovative solutions with major impact for the European economy and food additives production. The truly supra-disciplinary character of the Syncrop project offered unique inter-sectoral training for our ESRs and serves as career catalyst to enhance their employability: many ESRs who already earned the doctoral degrees have immediately found with new jobs, both in industry and academia, continuing to disseminate the know-how of SynCrop.
Our ESRs at the teaching module in Aberdeen 2019
Our ESRs at the teaching module in Vienna 2019
SynCrop Consortium at the midterm meeting in Delft 2019
Our ERSs at the teaching module in Vienna 2019