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Expanding the industrial use of Robust Oxidative Biocatalysts for the conversion and production of alcohols (ROBOX)

Periodic Reporting for period 3 - ROBOX (Expanding the industrial use of Robust Oxidative Biocatalysts for the conversion and production of alcohols (ROBOX))

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

Today’s quest for environmentally friendly and sustainable alternatives requires that traditional chemical processes be replaced by “greener” variants. Current industrial conversions are typically performed under harsh conditions, often requiring toxic chemicals and generating hazardous by-products. Enzymes have emerged as powerful alternatives to replace conventional chemical catalysts. This is because enzymes typically operate under mild reaction conditions. Moreover, enzyme-catalyzed reactions often display outstanding selectivity and good yield, which are difficult to achieve by chemical means. These features ensure that enzymes are also industrially relevant. The catalytic use of enzymes in chemical conversions is known as biocatalysis. Despite the many biocatalytic successes, full exploitation at an industrial scale is often hampered by the poor stability of many enzymes. A collection of robust enzymes is therefore a prerequisite to enable exploitation in industrial chemical conversions. The ROBOX project aimed at developing a set of stable oxidative enzymes and protocols on how to use them for industrial conversions. By targeting the potential of enzyme-based oxidations in conversions important to pharma, nutrition, fine and specialty chemicals as well as for materials, ROBOX greatly impacted the chemical industry as a whole as evidenced by the successfully executed demonstration cases.
ROBOX set out to demonstrate 11 target reactions for the markets mentioned above. The content of ROBOX was industry-driven with a final consortium made up of 17 partners that contained two major industrial companies and five SME’s. In addition to designing industrial biocatalytic processes, ROBOX also focused on technology development such as multi-parameter screening systems, computational methodologies and different microbial expression systems.
To conclude, an integrated approach was followed that involved the entire chain from enzyme discovery to industrial application. For the majority of the target reactions we developed suitable biocatalysts and could show their performance at an industrially relevant scale.
The research part of ROBOX was divided into five distinct but interrelated work packages.

The main objective of WP1 was to identify and engineer oxidative enzymes for the target reactions that were defined for the project. This included the development of new technology and new insights into enzyme functioning.
• The application of P450s is limited to very high value products such as drug metabolites that are of interest to the pharmaceutical industry.
• BVMOs have emerged as versatile and robust biocatalytic tools: shown to be relevant enzymes in the conversion of medium/low priced products.
• ADHs and AOXs were developed and successfully employed in several target reactions.

The main objective of WP2 was to identify the most competitive expression systems for ROBOX enzymes. For all ROBOX enzymes, robust, large-scale production procedures were developed. Effort was also spent on successfully developing standardized methods for the stabilization, storage and transport of enzymes produced in bacterial and fungal cells.

In WP3, the biocatalytic reactions targeted within ROBOX were designed and validated at small pilot scale. The main outcomes of WP3 are summarized below:
• P450s: Not all target reactions were achievable and these included the P450-catalyzed hydroxylation of aromatics. These conversions were therefore abandoned. It was found that P450s are suited for the production of high-value priced drug metabolites.
• BVMOs: These enzymes were applied in immobilized or free form together with a suitable cofactor regenerating enzyme to successfully develop biocatalytic processes at different scales for the conversion of cyclohexanone derivatives as well as macrocyclic and large cyclic ketones.
• AOX enzymes: a biocatalytic process for the conversion of vanillyl alcohol into vanillin was developed.
• ADHs: Promising enzymes from different ROBOX partners were used in immobilized or free form together with a suitable cofactor-recycling enzyme with great success to develop biocatalytic transformations for a range of stereo selective conversions.

WP4 concerned biocatalytic demonstration of ROBOX target reactions, resulting in: (1) process for the production of drug metabolites at 100 gram scale, (2) processes suitable for pharma, fine and specialty chemicals, nutritional, flavour & fragrance ingredients, and material science products in 10 – 200 liter reactors, (3) process for the production of monomers and novel polymers thereof on kg scale. Overall, it can be concluded that for all ROBOX enzymes (P450s, BVMOs, AOXs and ADHs) technically successful demonstrations were performed.

WP5 revolved around evaluation of the various case-studies (demonstrations) in ROBOX as well as an overall technology assessment. The overall evaluation of most case studies was very favorable, with a clear demonstration that the various techniques implemented in the project have worked. Benchmarking against chemical processes shows the enormous benefit of biocatalysis through reduction in reaction steps, thereby simplifying the production process.

WP6 and WP7 focused on dissemination, exploitation and management of the project. A proactive management approach was implemented and crucial. All objectives, tasks and deliverables as initially defined were continually implemented, monitored and updated to ensure all partners contributed to a comprehensive delivery.
An efficient collaboration resulted in high levels of engagement, dissemination and exploitation carefully linked to IP protection. The most important steps to take the work of ROBOX forward include: key results of the project were published in scientific journals, and major findings were patented by some of the industrial and academic partners. Other findings and novel technology originating from ROBOX will be further developed in other research projects and another result of the project is the start of a start-up biotech company.
ROBOX aimed at developing a set of stable oxidative enzymes and protocols on how to use them for industrial conversions. Therefore, an integrated approach was followed that involved the entire chain from enzyme discovery to application at a large scale. For most of the target enzymes/reactions, this strategy has worked successfully. For the majority of the >10 target reactions we developed a successful biocatalytic approach that is fit for industrial implementation. The research phase of ROBOX delivered a novel and unique set of robust oxidative enzymes that have emerged as promising biocatalytic tools.

The biocatalytic technology developed within the project significantly expands the toolkit of sustainable chemistry using oxidative enzymes. It can therefore be expected that the results of the project will contribute to the development of a biobased economy through, for example, production of “green” chemicals, such as: plastics, pharmaceuticals and inks. For one of the ROBOX target reactions we have performed a thorough comparative life cycle analysis that showed that the respective enzymatic oxidation has a lower environmental impact at laboratory scale when compared to its chemical equivalent if recycling of solvents and enzyme is considered. Moreover, the climate change impact of the biocatalytic reaction can be decreased by 75% when renewable electricity is used.
Reactions catalysed by the enzyme classes applied in ROBOX
Logo of the ROBOX project