Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages.

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in k(cat) for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions.