Engineering of unspecific peroxygenases for the β-hydroxylation of amines on a preparative scale.

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We are convinced that unspecific peroxygenases (UPOs) represent outstanding enzymes for C–H functionalizations with an extraordinary synthetic potential. By combining protein and process engineering, UPOs’ potential will be demonstrated by synthesizing pharmaceutically important building blocks on a gram-scale. UPOs are fungal enzymes that transfer one peroxide-borne oxygen to sp3-carbons. UPO-catalyzed reactions display an impressive substrate scope with more than 400 known examples. They exhibit excellent enantioselectivities and striking total turnover numbers of up to 300,000 for benzylic hydroxylations. Approx. four thousand putative UPO genes have been annotated, but less than 20 different UPO enzymes were studied in detail due to their challenging heterologous expression. These production limitations also significantly hampered targeted protein engineering efforts, and hence the current substrate scope consists primarily of wild-type activities. It would be of utmost importance to utilize UPOs’ catalytic machinery towards new industrially relevant substrates. In particular, substrates carrying aliphatic amines are ubiquitous in active pharmaceutical ingredients (API), but there are only scarce examples of UPOs hydroxylating these compounds. The molecular class of vicinal amino alcohols is of special interest as these groups could be synthesized by UPOs from amines and represent exciting scaffolds for the pharmaceutical industry. The proposed research project directly addresses the current limitations of UPOs towards amine substrates and aims to utilize an integrated approach of biochemical and process engineering towards the development and application of engineered UPOs. In the field of protein engineering, the methodology includes developing a rapid analysis system for the detection of amino alcohols and a re-shaping of the active site incl. amine anchored regions for the design of potent amine-converting UPOs. On the process engineering side, on-line methodologies for the control of hydrogen peroxide and integration of ion exchange and crystallization techniques will be tailor-made to fit the requirements of UPO-catalyzed reactions. Implementing the developed biocatalysts and methods will be investigated explicitly concerning a high substrate width and in gram-scale.The research project is organized into eight aims and four milestones, which will address the issue in a structured manner by both project partners. It starts from the developments and engineering of assays, proteins, real-time analysis, and selective product removals and culminates in preparative syntheses in gram-scale.