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BIOCASCADES- Sustainable and Scalable Biocatalytic Cascade Reactions Training Network

Periodic Reporting for period 2 - BIOCASCADES (BIOCASCADES- Sustainable and Scalable Biocatalytic Cascade Reactions Training Network)

Reporting period: 2017-01-01 to 2018-12-31

Cascade and one pot reactions represent an exciting recent development in White Biotechnology. The concept of performing multi-step syntheses in a concurrent fashion has received increased attention in the past years. Especially from a Green Chemistry point of view cascades represent a very promising approach, particularly due to the avoidance of intermediate downstream and purification steps. Such unit operation steps often contribute significantly to the overall environmental impact of a process. Furthermore, workup and isolation of intermediate products binds production capacities and resources, thus thereby also significantly contributing to production costs on industrial scale. ‘Smart’ cascades allow the utilization of a side product as cosubstrate for subsequent steps and thus allow substantial cost savings. Chemo-enzymatic cascade reactions are thus expected to make a major contribution to address one of the main challenges for the European industry, the development of sustainable and efficient production processes under the ‘green chemistry’ philosophy.

BIOCASCADES brought together a team of researchers from academia and industry for an interdisciplinary and intersectoral effort to overcome these challenges. It combined techniques from different scientific fields such as biocatalysis, transition-metal catalysis, compartmentalization, enzyme discovery, enzyme engineering and reaction engineering in order to develop commercially viable and environmentally benign chemo-enzymatic one pot reactions. The combination of different well established enzymes and chemical catalysts represents an innovative way to design novel cascades.
Scientific Objectives:
• To broaden the synthetic scope by combining enzymatic transformations with transition metal organic catalysts and to define strategies to increase the sustainability of these reactions.
• To use compartmentalization techniques and enzyme engineering to improve catalyst compatibility and suppress undesired cross reactivity.
• To optimize space-time yield and atom economy, by establishing novel methods for the shift of an equilibrium, such as in situ (co)-product removal, and by protein engineering.
• To develop strategies for the scale-up of viable chemo-enzymatic cascade reactions.

Addressing these challenges, within the project 11 early stage researchers were trained on the topic of chemo-enzymatic cascade reactions based on the following Training Objectives:
• Excellent research by developing novel chemo-enzymatic cascades for the production of complex amino alcohols and amines under the supervision of research leaders from biotechnology and chemistry.
• Interdisciplinarity and international networking to obtain all skills and knowledge required for the efficient combination of different chemical and biological catalysts in cascade reactions and their application (including scale-up) at companies. Learning to interact and communicate with scientists from different fields and sectors.
• Transferable skills and entrepreneurship by developing novel and robust processes under the supervision of founders of innovative companies. Anticipation of potential challenges in the early stages of planning and the development of entrepreneurial spirit and techniques how to overcome them, which is critical to bridge the innovation gap between academia and industry.
The doctoral research of 11 ESRs dealt with the development of (chemo-) enzymatic cascade reactions for the synthesis of optically pure amines and amino alcohols as pharmaceutical ingredients. Typically, two to three ESRs collaborated on a specific cascade reaction in order to approach challenges from different perspectives and to achieve a synergistic, multi-disciplinary, holistic proceeding. The intensive collaboration between academia and industry and the secondments of the ESRs to the laboratories of four SMEs and one large company allowed to consider crucial industrial aspects of the processes already at early stages of research.
Enzyme discovery and enzyme engineering were combined to develop catalysts with tailor-made properties for the special needs of the application in multi-enzyme cascade reactions. On the one hand, structure-inspired engineering of enzyme variants with improved stability allowed to widen the compatibility window of the catalysts in a cascade. On the other hand, targeted discovery of enzymes led to the identification of unique, tailor-made biocatalysts with suitable substrate spectra and selectivity to fulfill a particular role in enzyme cascade reactions.
After the initial proof-of-concept on small scale, the project also focused on reaction intensification and upscaling in an industrial environment. The upscaling revealed decisive challenges and bottlenecks. Based on a kinetic characterization of the different enzymes, optimal conditions for increasing substrate concentrations and the improvement of space-time-yields were identified. Immobilization of enzymes on solid carriers allowed to increase their stability. The latter was crucial to improve the overall productivity of enzyme cascades.
Within the project, two crucial parameters of cascade reactions could be successfully addressed: productivity and space-time-yield.
The BIOCASCADES project assembled a wide variety of different enzymes as diverse as omega-transaminases, alcohol dehydrogenases, heme-free mono- and dioxygenases, hydroxynitrile lyases, lipases, oxidases and halohydrin dehalogenases to seven new enzyme cascade reactions for the synthesis of optically pure amines and amino alcohols. Examples include both multi-enzyme and chemo-enzymatic cascades. Combining enzymatic transformations with transition-metal organic catalysts broadened significantly the catalytic scope of biocatalysis. Substrate loadings of up to 400 mM and concept studies in liter-scale demonstrated the usefulness of several cascades. The outcome of BIOCASCADES underlines the importance of a successful interaction of organic synthesis, biocatalysis and process intensification already at early stages of research and development.

The BIOCASCADES project impacted the involved researchers and institutions in several ways. One is the standardization of PhD requirements and standards among the different academic institutions. For the industrial institutions, the project gave them access to know-how of the academic environment in order to facilitate high quality PhD supervision and, at the same time, the academic institutions profited from intersectoral communication to obtain information on key aspects for industrial implementation of biocatalysis. The consortium provided a productive dynamic exchange of ideas in terms of scientific training and career development of the ESRs. The project partners disseminated the results in peer-reviewed publications and conference contributions. A conference organized by the project in Bielefeld with more than 80 participants brought together researchers from industry and academia to discuss research challenges and relevant scientific questions for the development of sustainable biocatalytic processes. Besides the various individual benefits for the ESRs, the intensive scientific exchange between the academic and industrial partners coming from different scientific fields also made this project highly fruitful for expanding the competencies as well as scientific network of the PIs, thus also contributing to their career development.
Biocascades Consortium Members and their expert contributions to the project