Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting

Abstract

De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as kcat/Km = 0.5 M(-1)s(-1) The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity.

Opportunities and challenges for combining chemo- and biocatalysis

“…In the search for these novel reactions catalysed by enzymes, various approaches have been utilized: de novo design of enzymes for novel activity, as pioneered by Baker (that is, Kemp eliminase, Diels-Alderase) is an important technique 71 . Although computationally designed NaTuRe CaTalysis enzymes are typically not highly active, subsequent evolution can boost their activity by multiple orders of magnitude and may lead to the creation of industrially relevant biocatalysts 72 . In addition, previously unobserved promiscuous activity of retro-aldolases has been reported, for example, variants of artificial aldolase RA95.5-8 catalysed the asymmetric synthesis of γ -nitroketones in a three-step, one-pot cascade 73 .…”

Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants

“…Searches of subspaces around such areas have repeatedly proven successful in enhancing protein functions and creating new activities. In protein engineering different approaches have been used ranging from introducing random mutations to the parent structure ( Copp et al , 2014 ) to recombination of related sequences by DNA shuffling ( Stemmer, 1994 ) or other methods ( Acevedo-Rocha et al , 2014 ; Obexer et al , 2016 ). When the 3D structure of the parent protein is known, mechanistic considerations and computational approaches can guide site-specific mutations, but the predictive power is still limited by incomplete understanding of transition states and reaction trajectories.…”

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

“…Furthermore, through the choice of appropriate intermediate mutants, experiments in droplets were steered towards a different evolutionary trajectory, identifying enzymes with ( S )‐enantioselectivity. In contrast, MTP‐based assays have only allowed access to ( R )‐enantioselective enzymes . Starting from the original computational design, a total of up to 10 8 protein variants have been screened.…”