Expanding the Enzyme Universe: Accessing Non‐Natural Reactions by Mechanism‐Guided Directed Evolution

Abstract

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe.

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

“…An improvement of these properties via this workflow sets the scene for step-by-step (45) accumulation of mutations that enhance evolvability (46,47) and allow adaptation of substrate specificity or introduction of mechanistic switches (48)(49)(50)(51). The more robust quadruple mutant PheDH V26I/N122S/L193M/T339I will be useful to buffer the effects of the growing mutational load that carries a stability cost in future evolution rounds.…”

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

“…The discovery [6][7][8] and engineering [9][10][11] of enzymes fueled by the formidable development in high-throughput biotechnology has allowed for tailored manufacturing of materials [12], medicines [13], and fine chemicals [14]. However, developing the full potential of biocatalysis and synthetic biology is dependent on exploring hitherto novel biochemistries [4,15] and reaction mechanisms [16] that expand the catalytic capabilities currently found in nature [15]. Interestingly, many enzymes display alternative chemistries and reaction mechanisms in addition to their natural substrate and reaction specificities [17].…”

“…Some prominent examples include the α,β-hydrolase fold [19] that is capable of harnessing 17 different reaction mechanisms [20], P450 monooxygenase-catalyzed cyclization [21], cyclopropanation [14], and γ-humulene synthase that generates 52 different natural products starting from the same polyisoprene substrate [22]. As accelerating promiscuous activities through enzyme engineering constitutes a cornerstone for expanding the catalytic scope of enzymes [15], an enhanced fundamental understanding of the molecular mechanisms underpinning the evolution of the catalytic function is desired. Atomistic details on the process by which protein sequence, structure, and function evolves has allowed researchers to predict enzyme activities in silico [23,24] and could furthermore illuminate how modern enzymes specialized upon diverging from ancient generalists.…”

Advantages of a distant cellulase catalytic base

“…Decomposition of the primary component, cellulose, is catalyzed by glycoside hydrolase (GH) enzymes, which are found ubiquitously in nature (2); therefore, improved catalytic efficiency of cellulose decomposition enzymes would help biomass to compete with non-renewable carbon sources. This motivates molecular-level studies into GH enzymatic mechanisms, as such understanding has previously proven invaluable in efforts to engineer variants with increased activities (3)(4)(5).A particularly important GH enzyme is Trichoderma reesei Cel6A (TrCel6A), which plays a key synergistic role in industrial enzyme cocktails for cellulose digestion. This enzyme is a cellobiohydrolase of GH family 6, which cleave β-1,4 glycosidic bonds processively along cellulose chains, from the non-reducing towards the reducing end, to release the glucose dimer cellobiose as the main product (6).…”