Creation of non-natural sugar enzymes by a conserved sequence-based recombination

Recent advances in directed evolution techniques including random mutagenesis and rational design have allowed us to engineer enzymes for broadening their industrial uses. However, such conventional approaches often require labor-intensive and complicated procedures to obtain tailor-made enzymes to meet industrial demands. Accordingly, we used a conserved sequence-based semi-rational approach using homologous sugar isomerases to engineer their enzymatic properties. We chose five homologs that have a variety of physicochemical properties for their activity and stability with respect to temperature and pH. Recombination of DNA fragments among those enzymes at their conserved regions yielded highly functional chimeras with unusual physicochemical properties, which were not found in parental wild-type enzymes. Detailed inspection of several chimeras demonstrated that our strategy can be very rapid and effective for elucidating structural and evolutionary features of sugar isomerases as well as finding their target regions to generate non-natural enzymes for industrial applications.