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Microbial deployment of new-to-nature chemistries for refactoring the barriers between living and non-living matter

Periodic Reporting for period 3 - MADONNA (Microbial deployment of new-to-nature chemistries for refactoring the barriers between living and non-living matter)

Período documentado: 2020-07-01 hasta 2022-06-30

The key challenge met by MADONNA was the programming of chemical reactions, whether biological or not, in order to eventually code them into DNA and implementing them in environmentally friendly and sustainable biological platfoms such as non-pathogenic microorganisms. This was pursued along the development of the Project by focusing the efforts around 4 objectives:

• Objective 1. Revisiting the Central Dogma, including the development of biological recipients to host reactions of industrial and environmental interest: CO2 fixation, dinitrotoluene (DNT) degradation and biological silicon-based metabolism.
• Objective 2. Upscaling the reactions in a usable bacterial chassis that can withstand the harsh conditions of either an industrial process or a variable physicochemical environment and becomes available in a readily operative format.
• Objective 3. Analysis of new to nature processes borne by the engineered agents by means of both á la carte theoretical frames and wet model systems to inspect the stability, efficacy and propagation potential of catalysts.
• Objective 4. In depth appraisal of the societal, economic and sustainability ramifications of NTN reactions pursued in the Project (and others in the future generated with identical technology) in the frame of the 4th Industrial Revolution.

These objectives align well with the necessity to tackle climate change. Whether mitigation or adaptation, the mainstream discourse thus far on how to deal with a warmer planet focuses on reducing emissions, finding incentives for reshaping/relocating economic activities and converting waste into value. These actions—even in the best-case scenario that they are generally implemented—are unlikely to produce the desired effects. The issue is therefore not so much to just moderate anthropogenic impacts but to foster conceptual and material tools for reverting pollution figures to pre-industrial times. MADONNA addresses these issues not only from a merely technical point, but also from a a wider social perspective and the ambition of involving all possible stakeholders.
Along the lifetime of the Project, a considerable number of important results and developments has materialized, including:

• The soil bacterium Pseudomonas putida has been upgraded as an optimal chassis for hosting environmentally significant new to nature reactions. This has involved a plethora of genetic tools for genome editing, containment for the sake of biosafety, production of chromosome-less vesicles and vectors for inter-species genome shuffling and transplantation. This works showcases the power of Synthetic Biology to deliver new materials and activities for the sake of industrial and environmental catalysis.

• An unprecedented progress has been made re. engineering and expression of entirely artificial metalloenzymes by capitalizing on the unique structure and properties of streptavidin. This enabled incorporation of [Fe4S4]-clusters and thus designing and evolving artificial metalloenzymes (ArM) for the reduction of CO2 to alka/enes (Fischer-Tropschase). These developments open immense perspectives of valorization of CO2 into added-value molecules.

• Procedures to synthesize organo-silicon compounds, although still in small amounts, have been developed and their incorporation to bacterial metabolic networks or biomass attempted. The wealth of knowledge on xenobiology accumulated along the way will be invaluable for future endeavours aimed at generating life forms that incorporate in their biomass a non-biological element and thus expand the scope of possible biochemistries in live systems.

• An entirely new concept for automatic and programmable chemical synthesis stemmed from earlier attempts to code-back reactions to DNA. This involves capturing the chemical synthesis literature from manual operation to a fully described and universal Chemical Description Language (χDL) to be run automatically in the Chemical Processing Unit, or ChemPU. This technology opens new perspectives for production of complex chemical structures.

• Novel models to predict risks of introducing new-to-nature reactions in the Biosphere with Synthetic Biology agents have fore the first time being produced. Such models exposed the limitations and opportunities of large-scale environmental interventions, in particular the containment that the naturally-occurring ecosystems impose to the spreading of new agents. Such a knowledge should improve the perception of risks associated to spreading of SynBio agents.

• Despite the COVID impasse, a strong dissemination / communication program was unfolded during the Project, including artists in Residence, exhibitions, videos aimed at making the MADONNA research and proposal of large-scale, SynBo-based environmental interventions acceptable by the general public and regulatory bodies with the basic idea of fostering a much needed revision of our mutuality with the natural world.
The starting point of MADONNA was the realization that reactions such as DNT dioxygenation by some bacteria interplayed directly with DNA because of their ability to generate reactive oxygen species (ROS). This produced genetic diversity and thus a superior ability to find viable outcomes to metabolic problems in a solution space. A separate approach involved design of altogether artificial enzymes based on implantation of reactive sites in otherwise non-active protein scaffolds—an strategy which has been particularly useful for development of new carboxylation processes able to remove and valorize CO2. Finally the Project addressed the complete coding and automation of chemical synthesis in a fashion reminiscent of the way DNA encodes complex metabolic processes in biological systems. Regardless of their origin (stress-created genetic diversification, rational enzyme design or robotically automated reactions) the ultimate fate of such processes was their implementation in natural or designer bacterial chassis able to self-replicate and deliver the corresponding activities when/where needed (e.g. industrial settings or given environments).

Biological coding á la carte of new-to-nature reactions in self-replicating hosts opens, on one hand amazing opportunities of environmentally-friendly and sustainable industries for the sake of the Circular Economy. But this is obviously not only a scientific or technical challenge, but one that has a large number of societal ramifications, which include not only the creation of an entirely new economic sector based on peribolism but also questions on ethical, security, safety, economic, governance and public perceptions aspects. These aspects have but intensified during the last period of the Project lifetime due to the growing awareness of climatic change and the need to develop technologies to first mitigate and ultimately revert such a change. In this sense, MADONNA has seriously contributed to move the global discussion on mitigation and adaptation to climate change towards the need to develop technologies and models for large-scale enviromental interventions. In fact, developing the theoretical and practical tools to deal with such a phenomenal challenge: the deliberate engineering of a new connectivity between industrial processes and the biogeochemical courses of the Biosphere that creates new elementary cycles and enhances performance and sustainability of the industrial metabolism.
Biological metabolism versus industrial metabolism
Canonical and non-canonical coding of new reactions in DNA
Synthetic Biology enable the design of agents able to deliver new-to-nature-reactions
Breaking the boundaries of biological vs chemical evolution
Human impact makes ecosystems to run into a non-reversible tipping point