Neutral Genetic Drift-Based Engineering of a Sucrose-Utilizing Enzyme toward Glycodiversification

Abstract

Neutral drift (also called purifying selection) is an attractive approach to generate polymorphic variant libraries for enzyme engineering. Here, we have applied this strategy to modify the substrate specificity of a transglucosylase. Our model enzyme, the amylosucrase from Neisseria polysaccharea, is a glucosylation biocatalyst of prime interest because it uses the widespread substrate sucrose as a glucosyl donor and shows broad acceptor promiscuity. A library of 440 functional amylosucrase variants was generated after four rounds of neutral drift at a low mutation rate. The functional variations present in this library were investigated by assaying the ability of these variants to use an alternative glucosyl donor (p-nitrophenyl-α-d-glucopyranoside, pNP-Glc) and to glucosylate a range of acceptors (including methyl-α-l-rhamnopyranoside, which is not naturally recognized by the parental enzyme). The impact of these mutations on the thermal stability of the variants was also assessed. Large variations of acceptor promiscuity were observed, ranging from the complete loss of detectable activity to a 2-fold increase relative to the parental enzyme. Variants showing increased catalytic efficiency toward the alternative pNP-Glc donor were also identified. Specifically, one variant combining four unprecedented amino acid changes was 25-fold more efficient at utilizing pNP-Glc than the parental enzyme and acquired glucosylation activity toward methyl-α-l-rhamnopyranoside. Enzymes with improved thermal stability were also identified. Overall, our work demonstrates that neutral drift is an effective and powerful strategy to engineer transglycosylases with enhanced or even acquired substrate specificities from small-sized functional libraries compatible with accurate low-throughput multi-parameter analyses.