# Studying the evolutionary potential of ancestral aryl sulfatases in the alkaline phosphatase family with droplet microfluidics

## TL;DR

This paper uses droplet microfluidics to study how ancestral and modern aryl sulfatase enzymes evolve and respond to mutations.

## Contribution

The study introduces ultrahigh-throughput droplet microfluidics to compare ancestral and extant aryl sulfatases and their evolvability.

## Key findings

- Higher mutation rates led to a wider distribution of active enzyme variants but fewer overall improvements.
- Different enzymes responded variably to mutation rates, highlighting the need for diverse mutagenesis strategies.
- Droplet microfluidics enabled efficient screening of enzyme activity and recovery of improved variants.

## Abstract

Characterizing the dynamics and functional shifts during protein evolution is essential, both for understanding protein evolution and for rationalizing efficient strategies for e.g. enzymes with desired and effective functions. Most proteins organize in families, sets of divergent sequences which share a common ancestor and have a similar structural fold. Here, we study aryl sulfatases, a subfamily of the large and evolutionary old alkaline phosphatase superfamily. We demonstrate how ultrahigh-throughput droplet microfluidics can be used for studying aryl sulfatases and their computationally reconstructed putative common ancestors. We compare the evolvability and robustness of three ancestors and three extant aryl sulfatases which all exhibit catalytic promiscuity towards a range of substrate classes. Using varying mutations rates, eleven libraries were constructed and expressed in single-cell microdroplets. In general, higher mutation rates resulted in wider distribution of active variants but fewer improved variants overall. However, the impact of mutation rate differed between enzymes, with some benefiting from higher and others from lower mutation rate, underscoring the need to test diverse mutagenesis regimes.

Extant and reconstructed ancestral aryl sulfatases were compared using directed evolution. Mutant libraries were screened using microfluidic droplet sorting, the activity of each variant was measured and improved variants were recovered.