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Rules and Tools for Efficient Enzyme Evolution, Recruitment and Discovery Based on Catalytic Promiscuity
‘Promiscuous’ enzymes possess additional activities in addition to their native ones, challenging the textbook adage “one enzyme – one activity”. The observation of strong promiscuous activities in the alkaline phosphatase (AP) superfamily - where one active site can catalyse up to six chemically distinct hydrolytic reactions with promiscuous second order rate accelerations between 10⁹ and 10¹⁷ - suggests that even broadly promiscuous catalysis can be rather efficient. We demonstrate by directed evolution and phylogenetic analysis that crosswise promiscuity relationships in the AP superfamily indicates that an enzyme is ‘pregnant’ with another activity, i.e. has the potential to be mutated or evolved into a new catalyst. These catalysts are multifunctional generalists that have won additional activities, at varying trade-off cost to the other existing activities. The systematic comparative analysis promiscuous relationships in enzyme superfamilies on the level of structure, sequence similarity, specificity and reactivity suggests factors that govern evolutionary adaptation. To efficiently explore the interconversion of promiscuous enzyme, we use picoliter water-in–oil emulsion droplets produced in microfluidic devices as high-throughput screening reactors. We present new workflows that allow screening of >10⁶ clones and allows successful selections from single protein and metagenomic libraries, where lower throughput approaches have failed.