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Sustainable industrial processes based on a C-C bond-forming enzyme platform

Periodic Reporting for period 3 - CARBAZYMES (Sustainable industrial processes based on a C-C bond-forming enzyme platform)

Reporting period: 2018-04-01 to 2019-03-31

Large-scale industrial production of chemicals often takes place with reduced efficiency and produces large quantities of toxic waste. Improved biocatalysts for carbon-carbon bond formation such as those developed within the CarbaZymes project can lead to novel, safer and more efficient industrial processes, which will benefit industry, consumers and the environment.
Products of industrial organic synthesis such as pharmaceuticals and bulk chemicals contribute significantly to the quality of life and the economic development of modern societies. However, industrial chemical processes carried out through “classic” chemistry and catalysis often involve the use of hazardous substances and considerable amounts of energy. By contrast, enzymes are natural catalysts that speed up the manufacture and modification of molecules under mild reaction conditions and with exquisite selectivity, leading to increased product yields. Thus, the application of enzymes (“biocatalysis”) to catalyze industrial reactions offers great potential to improve manufacturing processes by reducing the use of corrosive chemicals, organic solvents, toxic metals and energy, in compliance with the concept and principles of Green Chemistry.
Carbon-carbon bond forming reactions (carboligation) are key processes in industrial organic synthesis, to create innovative molecules of high value, but remain insufficiently explored also due to the lack of robust enzymes that cover a suitably wide range of reactions. To bridge the gap between laboratory research and industrial large-scale production, the scientists behind CarbaZymes addressed these challenges by developing a broad toolbox of carbon-carbon bond forming enzymes tailored to industrial needs: the consortium applied cutting-edge molecular biology methods in combination with specifically developed software (exclusivity of one of the consortium´s industrial partners) to selectively improve their properties so that these enzymes can be efficiently applied in novel, sustainable industrial processes with reduced environmental footprints. The CarbaZymes project pursued the biocatalytic synthesis of several valuable compounds, spanning a range of four chiral fine chemicals and three bulk chemicals, corresponding to significant market needs in the chemical, biotechnological and pharmaceutical industries.
The overall objective of the CarbaZymes project was to generate a platform of robust enzymes for diverse types of carboligation reactions, and to demonstrate their potential for large-scale biocatalytic synthesis under industrial constraints. In its work program, the consortium has successfully established advanced tools, methods and patented processes for the industrial synthesis of the targeted compounds and has developed and produced a broad panel of improved enzymes for carbon-carbon bond formation. The toolbox of methodologies includes new bioinformatics tools, tests for high-throughput reaction analysis, protein structure modeling, as well as ultra-fast protocols for enzyme engineering and enzyme stabilization.
A bioinformatics system was set up to facilitate in-depth data mining for several target enzyme superfamilies to aid in the discovery and development of novel biocatalysts. Structural models were built to support a deeper understanding of structure-function relationships within the known natural variety. Enzyme thermostabilization methods were also developed and successfully implemented. Using these advanced tools, the CarbaZymes project produced several superfamily libraries of novel enzymes, which were subsequently tested against a collection of known enzymes, for their performance characteristics in various reactions within the scope of the project. Among the expanded panels of novel biocatalysts, several key enzymes could be identified that show significantly better properties. These enzymes are suitable for assembly into efficient catalytic reaction cascades for the synthesis of the targeted molecules, and for further developments beyond the project’s goals. Libraries of mutant enzymes were constructed, in a search for catalysts with improved properties for the target reactions by in-vitro-evolution and high-throughput screening. Several engineered enzymes were identified and used to catalyze highly selective conversions. These gave access to novel chiral building blocks, potentially valuable for industrial production, which were not accessible previously. Novel total synthesis protocols were developed which gave rise to high volumetric productivity for the synthesis of a broadly used amino acid and an API precursor for much needed pharmaceuticals. Modeling of reaction systems for larger scale carboligation processes was carried out, to enable the development of scalable biocatalytic syntheses. Microreactor and large-scale bio-reactor technology has been set up for bioprocess characterization and advanced reaction engineering of selected processes. Industrial implementation of the research´s findings has been conducted successfully at pilot-plant level for evaluation of its economic feasibility at a larger scale. Patent applications have been filed to secure intellectual property rights.
Placing a strong emphasis on dissemination, the CarbaZymes consortium partners actively engaged in communicating new discoveries and the knowledge gained to the scientific community and the wider public. This included 22 scientific publications, several workshops and practical training courses, participation in key biotechnology conferences and the creation of two CarbaZymes videos available on YouTube:

Moreover, promotion of White Biotechnology was carried out via networking events and contacts to the general public through web presence, newsletters and social media channels. A major scientific dissemination activity was the organization of the international outreach conference “Novel Enzymes 2018” in Darmstadt, Germany, and the co-organization — with project ROBOX — of a H2020 Demonstration Workshop “New developments in industrial biocatalysis” in Frankfurt, Germany.
The CarbaZymes outcomes will have a notable social and economic impact, by addressing the pressing needs of an increasing and aging population, by addressing billion-Euro markets and by creating new intellectual property. CarbaZymes aimed to promote innovation in the field of carbon-carbon bond formation at large scale, and achieved this by evaluating a vast number of novel enzymes for carbon-carbon bond formation, and by developing natural enzyme-catalyzed routes to valuable specialty and bulk chemical precursors of great significance and economic value.
The extended range of novel and improved biocatalysts generated from this research will provide wide-ranging opportunities for the synthesis of many additional valuable products, thus making a profound impact on reducing emissions, energy consumption and toxic waste. By developing new intellectual property as well as advancing biotechnology know-how — a Key Enabling Technology for Europe — this project strengthens the global competitiveness of the European chemical, biotechnological and pharmaceutical industries. Moreover, it provided state-of-the-art training and generated many highly skilled researchers who will drive European industrial innovation while improving resource efficiency.
The CarbaZymes logo
1st CarbaZymes newsletter cover highlighting chiral C-C bond formation