Periodic Reporting for period 1 - RADICALZ (RAPID DISCOVERY AND DEVELOPMENT OF ENZYMES FOR NOVEL AND GREENER CONSUMER PRODUCTS)
Reporting period: 2021-06-01 to 2022-11-30
The current climate and sustainability crises represent a risk to health and life, emphasising the need for an accelerated transition towards a circular bioeconomy as part of a general framework of a greener and more competitive industry. The application of enzymes in industrial processes is increasingly important to achieve the EU’s sustainability goals and strengthen the circular bioeconomy, because of their capacity to transform (bio)molecules selectively and efficiently. Thus, enzymes help enable the replacement of oil-based chemistry for the use bio-based feedstocks, preferably from secondary waste streams, thus transitioning from linear to circular consumption models.
However, enzymes still find hurdles for their industrial application: low success rates of discovery and engineering; tedious and expensive methods to explore diversity; and limited activity/stability in the final application. RADICALZ assembles an interdisciplinary and intersectoral consortium to deliver faster, more versatile and more affordable tools for enzyme discovery and engineering, enabling the development of novel enzymes, new formulations and ingredients for more environment-friendly and healthier consumer products in a timeframe compatible with industrial development. This project will: i) develop new droplet microfluidic tools to find suitable enzymes for consumer products; ii) develop user-friendly software solutions based on machine learning (ML) for faster and more accurate enzyme engineering; iii) develop novel enzymes and bio-based, bio-catalytically synthesized ingredients for consumer products (glycosides, wash-enhancing enzymes, bio-based thickeners, natural antioxidants and fragrances); iv) develop bio-based, condition-responsive capsules for the protection and triggered release of enzymes and ingredients in the formulation of consumer products.
However, enzymes still find hurdles for their industrial application: low success rates of discovery and engineering; tedious and expensive methods to explore diversity; and limited activity/stability in the final application. RADICALZ assembles an interdisciplinary and intersectoral consortium to deliver faster, more versatile and more affordable tools for enzyme discovery and engineering, enabling the development of novel enzymes, new formulations and ingredients for more environment-friendly and healthier consumer products in a timeframe compatible with industrial development. This project will: i) develop new droplet microfluidic tools to find suitable enzymes for consumer products; ii) develop user-friendly software solutions based on machine learning (ML) for faster and more accurate enzyme engineering; iii) develop novel enzymes and bio-based, bio-catalytically synthesized ingredients for consumer products (glycosides, wash-enhancing enzymes, bio-based thickeners, natural antioxidants and fragrances); iv) develop bio-based, condition-responsive capsules for the protection and triggered release of enzymes and ingredients in the formulation of consumer products.
In this reporting period (M1-M18), RADICALZ has selected candidate enzymes as ingredients in “smart” consumer products as well as for syntheses of bio-based ingredients for laundry detergents, nutraceuticals and cosmetics. Specifically, our targets include enzymes for washing powders, glycoside-synthesising enzymes and diverse enzymes for biocatalytic transformations of bio-based feedstocks into added value antioxidants, emulsifiers, thickeners, aromas. The Consortium’s vast resources, including large collections, metagenomes acquired in past EU-funded actions and public databases, constitute a priceless asset for novelty, above all when combined with adequate, generalist and fast tools for in silico or in vitro mining of sequence space, such as the proprietary 3DM systems of BIOP, sequence similarity network (SSN)-based mining at BRAIN and INSAT or the microfluidic workflows developed at UNEXE, UAM and INSAT. In particular, SSNs to reveal novel regions of sequence (and likely, functional) space has been leveraged for enzymes belonging to the transferase, hydrolase and lyase classes, reinforcing the general validity of this approach. Also, the versatility of droplet microfluidics for enzyme screening and formulation has been illustrated by developing bespoke droplet workflows to determine synthesis and degradation of polymers, assays with ca. single-molecule sensitivity as well as droplet-based capsules for enzyme and microbial biotransformations.
Finally, environmental metrics as well as process modelling are being used project-wide to pinpoint potential pitfalls of the envisioned processes. The former has outlined the critical relevance of process intensification and the nature and treatment of the reaction medium turned into wastewater after the reaction (the downstream). These general guidelines will be further customised once optimised enzymes and reaction conditions are available for each of the target processes. Regarding the latter, three different processes have been modelled in detail, uncovering substrate and product inhibitions that will result in the substitution of enzymes or changes in the substrate feeding regimes. Therefore, we have illustrated the general validity of the diagnostic tools of RadicalZ, similarly to the enzyme discovery tools.
As a result, suitable candidate enzymes have been found for almost all of the envisioned processes, including several biotransformations described herein for the first time. Consequently, most of the project has moved into the protein or reaction engineering phase with the selected candidate enzymes.
Finally, environmental metrics as well as process modelling are being used project-wide to pinpoint potential pitfalls of the envisioned processes. The former has outlined the critical relevance of process intensification and the nature and treatment of the reaction medium turned into wastewater after the reaction (the downstream). These general guidelines will be further customised once optimised enzymes and reaction conditions are available for each of the target processes. Regarding the latter, three different processes have been modelled in detail, uncovering substrate and product inhibitions that will result in the substitution of enzymes or changes in the substrate feeding regimes. Therefore, we have illustrated the general validity of the diagnostic tools of RadicalZ, similarly to the enzyme discovery tools.
As a result, suitable candidate enzymes have been found for almost all of the envisioned processes, including several biotransformations described herein for the first time. Consequently, most of the project has moved into the protein or reaction engineering phase with the selected candidate enzymes.
• The tools, enzymes, processes and molecules developed in the first reporting period and those under ongoing development build on previous developments of RadicalZ partners, who represent the state-of-the art in their respective disciplines. As a non-comprehensive summary, RADICALZ has gone beyond the state of the art by:
• demonstrating the validity of sequence similarity networks to pinpoint novel regions of sequence space for several of the enzyme classes of the project. Representatives of these regions are expected to contain new functionalities and new sequences, and therefore a higher likelihood of novel IP and ultimately innovative value for the industrial partners of RADICALZ.
• developing 2 novel enzyme assays and workflows in droplet format to speed up enzyme discovery 1000x and reduce assay costs by a million-fold. These will result in the reduction of costs to develop/discover novel enzymes, in turn impacting on the reduction of costs of the biocatalytically synthesized molecules and ultimately of consumer products.
• demonstrating the feasibility of previously undescribed glycosylation reactions, including the modification of bulky molecules and polyphenols. This will result in greener, more sustainable consumer products, whose ingredients are biosourced, thus contributing to sustainability and a circular bioeconomy
• Innovative ML tools developed in this project, which build on previous know-how of partner BIOP and which outperform all benchmarked tools. These tools are the seed of AI-guided protein engineering, to increase the efficiency, reduce the time needed to improve proteins for industrial application, and ultimately democratize protein engineering.
• Recommendations for greener biocatalytic processes based on a thorough analysis of the upstream, reaction and downstream processing. These recommendations are specific for RADICALZ reactions, but can be modified to fit other biocatalytic processes and therefore constitute a roadmap for process development that may feed policy recommendations.
⦁ Analysis tools that optimize the communication and dissemination of the project over social media, including expanding the range of stakeholders that the project can reach. Ultimately, the use of these tools results in enhanced impact of the project, maximising the return on investment in research and innovation, and promoting the effort of the European Commission towards a resilient, competitive and sustainable European economy.
• demonstrating the validity of sequence similarity networks to pinpoint novel regions of sequence space for several of the enzyme classes of the project. Representatives of these regions are expected to contain new functionalities and new sequences, and therefore a higher likelihood of novel IP and ultimately innovative value for the industrial partners of RADICALZ.
• developing 2 novel enzyme assays and workflows in droplet format to speed up enzyme discovery 1000x and reduce assay costs by a million-fold. These will result in the reduction of costs to develop/discover novel enzymes, in turn impacting on the reduction of costs of the biocatalytically synthesized molecules and ultimately of consumer products.
• demonstrating the feasibility of previously undescribed glycosylation reactions, including the modification of bulky molecules and polyphenols. This will result in greener, more sustainable consumer products, whose ingredients are biosourced, thus contributing to sustainability and a circular bioeconomy
• Innovative ML tools developed in this project, which build on previous know-how of partner BIOP and which outperform all benchmarked tools. These tools are the seed of AI-guided protein engineering, to increase the efficiency, reduce the time needed to improve proteins for industrial application, and ultimately democratize protein engineering.
• Recommendations for greener biocatalytic processes based on a thorough analysis of the upstream, reaction and downstream processing. These recommendations are specific for RADICALZ reactions, but can be modified to fit other biocatalytic processes and therefore constitute a roadmap for process development that may feed policy recommendations.
⦁ Analysis tools that optimize the communication and dissemination of the project over social media, including expanding the range of stakeholders that the project can reach. Ultimately, the use of these tools results in enhanced impact of the project, maximising the return on investment in research and innovation, and promoting the effort of the European Commission towards a resilient, competitive and sustainable European economy.