Enriching productive mutational paths accelerates enzyme evolution.

Darwinian evolution has given rise to all the enzymes that enable life on Earth. Mimicking natural selection, scientists have learned to tailor these biocatalysts through recursive cycles of mutation, selection and amplification, often relying on screening large protein libraries to productively modulate the complex interplay between protein structure, dynamics and function. Here we show that by removing destabilizing mutations at the library design stage and taking advantage of recent advances in gene synthesis, we can accelerate the evolution of a computationally designed enzyme. In only five rounds of evolution, we generated a Kemp eliminase-an enzymatic model system for proton transfer from carbon-that accelerates the proton abstraction step >108-fold over the uncatalyzed reaction. Recombining the resulting variant with a previously evolved Kemp eliminase HG3.17, which exhibits similar activity but differs by 29 substitutions, allowed us to chart the topography of the designer enzyme's fitness landscape, highlighting that a given protein scaffold can accommodate several, equally viable solutions to a specific catalytic problem.

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis.

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants.

LibGENiE - A bioinformatic pipeline for the design of information-enriched enzyme libraries.

Enzymes are potent catalysts with high specificity and selectivity. To leverage nature's synthetic potential for industrial applications, various protein engineering techniques have emerged which allow to tailor the catalytic, biophysical, and molecular recognition properties of enzymes. However, the many possible ways a protein can be altered forces researchers to carefully balance between the exhaustiveness of an enzyme screening campaign and the required resources. Consequently, the optimal engineering strategy is often defined on a case-by-case basis. Strikingly, while predicting mutations that lead to an improved target function is challenging, here we show that the prediction and exclusion of deleterious mutations is a much more straightforward task as analyzed for an engineered carbonic acid anhydrase, a transaminase, a squalene-hopene cyclase and a Kemp eliminase. Combining such a pre-selection of allowed residues with advanced gene synthesis methods opens a path toward an efficient and generalizable library construction approach for protein engineering. To give researchers easy access to this methodology, we provide the website LibGENiE containing the bioinformatic tools for the library design workflow.

Engineering Fe(II)/α-Ketoglutarate-Dependent Halogenases and Desaturases.

Fe(II)/α-ketoglutarate-dependent dioxygenases (α-KGDs) are widespread enzymes in aerobic biology and serve a remarkable array of biological functions, including roles in collagen biosynthesis, plant and animal development, transcriptional regulation, nucleic acid modification, and secondary metabolite biosynthesis. This functional diversity is reflected in the enzymes' catalytic flexibility as α-KGDs can catalyze an intriguing set of synthetically valuable reactions, such as hydroxylations, halogenations, and desaturations, capturing the interest of scientists across disciplines. Mechanistically, all α-KGDs are understood to follow a similar activation pathway to generate a substrate radical, yet how individual members of the enzyme family direct this key intermediate toward the different reaction outcomes remains elusive, triggering structural, computational, spectroscopic, kinetic, and enzyme engineering studies. In this Perspective, we will highlight how first enzyme and substrate engineering examples suggest that the chemical reaction pathway within α-KGDs can be intentionally tailored using rational design principles. We will delineate the structural and mechanistic investigations of the reprogrammed enzymes and how they begin to inform about the enzymes' structure-function relationships that determine chemoselectivity. Application of this knowledge in future enzyme and substrate engineering campaigns will lead to the development of powerful C-H activation catalysts for chemical synthesis.

Directed evolution of carbon-hydrogen bond activating enzymes.

As industrial biocatalysis is maturing, access to enzymatic activities beyond chiral resolutions, asymmetric ketone reductions and reductive aminations is gradually becoming reality. Especially the utilization of carbon-hydrogen bond (C-H) activating enzymes is very attractive as they catalyze a variety of chemically extremely challenging transformations. Because of their intrinsic complexity, the use of these enzymes in manufacturing has been limited. However, recent advances in enzyme engineering and bioinformatics have led to activity improvements for native and non-native substrates, the introduction of new-to-nature chemistries and the identification of promising novel enzyme families. Looking forward, the use of automation and advanced computer algorithms will help to streamline the evolution process of C-H activating enzymes leading to more robust and active biocatalysts.