Berndjan Eenink (IEB WWU)
in: 'Frontiers in Ecology and Evolution': "Probing the evolvability of ancestral and extant sulfatases using fluorescence-activated droplet sorting (FADS)"
by Berndjan Eenink (IEB WWU)
Abstract: We investigate the effect of mutational load on extant and reconstructed ancestral members of the Alkaline Phosphatase (AP) superfamily to study the shift in primary and secondary activities along the evolutionary part back to their last common ancestor. We are interested both in understanding natural protein evolution and how to harness our knowledge of natural evolution to devise improved strategies for artificial laboratory evolution. The combination of mutations needed for desired improvements may not be reachable in one-by-one mutational steps due to the occurrence of epistatic ratchets, raising the question if and how evolutionary dead ends can be bypassed. For identifying an ideal starting point for laboratory evolution, the shape of the local fitness landscape could be even more critical than the initial activity levels of an enzyme. We developed a novel method to sort sulfates displayed on the surface on E. coli in a high-throughput fashion using fluorescence-activated droplet sorting (FADS) individual cells are encapsulated into picolitre volume microdroplets and sorting based on fluorescence level, which was used to successfully evolve sulfatase SpAS1. Since then we have developed a method to grow cells inside picolitre volume droplets prior to expression and sorting. Individual cells are encapsulated with autoinduction medium. After overnight incubation E. coli cells divide inside the droplets, resulting in multiple copies harboring identical enzyme variants inside each droplet. With this droplet-growth method we have screened libraries of four ancestral and four extant aryl sulfatases in order to compare the evolvability of ancestral and extent sulfatases.