Exploration of Metagenomic Racemases for Biosynthetic Cascades

Biocatalysis becomes more and more appealing to industry due to its inherent ability to address contemporary demands for ecological, stereoselective synthesis from benign and biologically sourced materials, like amino acids. This creates an endless desire for new, diverse enzyme catalysts. One of the advantages of biocatalysis is to inherently provide catalysts for dynamic kinetic resolution like amino acid racemases, which is key to economic stereoselective synthesis. Drawback of most known amino acid racemases is their specificity for only few amino acids. Therefore, this work is dedicated to finding new and unusual amino acid racemases within a library that was created by our industry partner Prozomix Ltd. from their metagenomic database. Besides discovering enzymes with broader, or rare reactivities, the goal was also to demonstrate their synthetic potential in biocatalytic one-pot sequential cascade reactions. First step was to adapt a spectrophotometric assay to enable efficient probing of the 56 library enzymes for activity on 16 canonical and 14 selected non-canonical amino acids. In addition, the enzymes’ melting points were measured with nanoDSF to assess their thermal stability. Subsequently, biocatalytic reaction cascades were implemented. For the production of the rare sugars 5-deoxy- and 1,5-dideoxyfructose, the detected racemase-hits were combined with D-amino acid oxidase and transketolase, and comparing this oxidase pathway with an alternative transaminase pathway. Valuable 2-keto acids, and D-2-hydroxy acids were produced, employing selected racemases together with oxidase and stereoselective dehydrogenase enzymes. Moreover, new techniques were developed to improve one of these cascades’ critical sub-process, the provision of oxygen to the oxidase at laboratory-scales. Required substrates for screening and sugar-producing cascades were synthesized, optimizing existing protocols and developing new, improved routes and methodologies. Several racemases with remarkable substrate ranges were discovered, for example enzymes racemising tyrosine, or acting on the dipeptide β-aspartame. Some of the panel enzymes show both, broad substrate range and high thermostability of Tm > 69 °C. Their synthetic potential was successfully demonstrated by producing rare sugars and amino acid derivatives with productivities of up to 15.3 g D-phenyllactic acid per gram of cell-free racemase extract. Thus, the research goal to discover useful, innovative racemases, fit for synthetic application in dynamic kinetic resolutions was achieved and these novel enzymes may even serve as starting points for enzyme engineering.