Improved biocatalysts from a synthetic circular permutation library of the flavin-dependent oxidoreductase old yellow enzyme.

Members of the old yellow enzyme (OYE) family are widely used, effective biocatalysts for the stereoselective trans-hydrogenation of activated alkenes. To further expand their substrate scope and improve catalytic performance, we have applied a protein engineering strategy called circular permutation (CP) to enhance the function of OYE1 from Saccharomyces pastorianus. CP can influence a biocatalyst's function by altering protein backbone flexibility and active site accessibility, both critical performance features because the catalytic cycle for OYE1 is thought to involve rate-limiting conformational changes. To explore the impact of CP throughout the OYE1 protein sequence, we implemented a highly efficient approach for cell-free cpOYE library preparation by combining whole-gene synthesis with in vitro transcription/translation. The versatility of such an ex vivo system was further demonstrated by the rapid and reliable functional evaluation of library members under variable environmental conditions with three reference substrates ketoisophorone, cinnamaldehyde, and (S)-carvone. Library analysis identified over 70 functional OYE1 variants with several biocatalysts exhibiting over an order of magnitude improved catalytic activity. Although catalytic gains of individual cpOYE library members vary by substrate, the locations of new protein termini in functional variants for all tested substates fall within the same four distinct loop/lid regions near the active site. Our findings demonstrate the importance of these structural elements in enzyme function and support the hypothesis of conformational flexibility as a limiting factor for catalysis in wild type OYE.