Unspecific peroxygenases (UPOs) are fungal enzymes that attract significant attention for their ability to perform versatile oxyfunctionalization reactions using H2O2. Unlike other oxygenases, UPOs do not require additional reductive equivalents or electron transfer chains that complicate basic and applied research. Nevertheless, UPOs generally exhibit low to no heterologous production levels and only four UPO structures have been determined to date by crystallography limiting their usefulness and obstructing research. To overcome this bottleneck, we implemented a workflow that applies PROSS stability design to AlphaFold2 model structures of 10 unique and diverse UPOs followed by a signal peptide shuffling to enable heterologous production. Nine UPOs were functionally produced in Pichia pastoris, including the recalcitrant CciUPO and three UPOs derived from oomycetes the first nonfungal UPOs to be experimentally characterized. We conclude that the high accuracy and reliability of new modeling and design workflows dramatically expand the pool of enzymes for basic and applied research.

Unspecific peroxygenases (UPOs) perform oxy-functionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, β-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC−MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-β-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively.

Engineering proteins and enzymes with the desired functionality has broad applications in molecular biology, biotechnology, biomedical sciences, health, and medicine. The vastness of protein sequence space and all the possible proteins it represents can pose a considerable barrier for enzyme engineering campaigns through directed evolution and rational design. The nonlinear effects of coevolution between amino acids in protein sequences complicate this further. Data-driven models increasingly provide scientists with the computational tools to navigate through the largely undiscovered forest of protein variants and catch a glimpse of the rules and effects underlying the topology of sequence space. In this review, we outline a complete theoretical journey through the processes of protein engineering methods such as directed evolution and rational design and reflect on these strategies and data-driven hybrid strategies in the context of sequence space. We discuss crucial phenomena of residue coevolution, such as epistasis, and review the history of models created over the past decade, aiming to infer rules of protein evolution from data and use this knowledge to improve the prediction of the structure− function relationship of proteins. Data-driven models based on deep learning algorithms are among the most promising methods that can account for the nonlinear phenomena of sequence space to some degree. We also critically discuss the available models to predict evolutionary coupling and epistatic effects (classical and deep learning) in terms of their capabilities and limitations. Finally, we present our perspective on possible future directions for developing data-driven approaches and provide key orientation points and necessities for the future of the fast-evolving field of enzyme engineering.

A protocol for synthesizing triazole-containing pyrazolines and pyrazoles selectively using trifluoromethylated 5-(1,2,3-triazol-1-yl)enones as starting materials, is reported. The selectivity of the reaction was controlled by the nature of the hydrazine or derivative used: free hydrazines furnished the 1,5-regiosiomer exclusively in yields up to 98%, whereas protected hydrazines provided the 1,3-regioisomer in yields up to 77%. To demonstrate the synthetic versatility of the triazole-based enone, reactions with other unsymmetrical dinucleophiles (hydroxylamine hydrochloride and S-methyl isothiourea sulfates) allowed the selective preparation of triazole-containing isoxazoline and pyrimidine rings.

In recent years, the engineering of flexible loops to improve enzyme properties has gained attention in biocatalysis. Herein, we report a loop engineering strategy to improve the stability of the substrate access tunnels, which reveals the molecular mechanism between loops and tunnels. Based on the dynamic tunnel analysis of CYP116B3, five positions (A86, T91, M108, A109, T111) in loops B-B′ and B′-C potentially affecting tunnel frequent occurrence were selected and subjected to simultaneous saturation mutagenesis. The best variant 8G8 (A86T/T91L/M108N/A109M/T111A) for the dealkylation of 7-ethoxycoumarin and the hydroxylation of naphthalene was identified with considerably increased activity (134-fold and 9-fold) through screening. Molecular dynamics simulations showed that the reduced flexibility of loops B-B′ and B′-C was responsible for increasing the stability of the studied tunnel. The redesign of loops B-B′ and B′-C surrounding the tunnel entrance provides loop engineering with a powerful and likely general method to kick on/off the substrate/product transportation.

Enzymatic hydroxylation of activated and nonactivated sp3-carbons attracts keen interest from the chemistry community as it is one of the most challenging tasks in organic synthesis. Nature provides a vast number of enzymes with an enormous catalytic versatility to fulfill this task. Given that those very different enzymes have a distinct specificity in substrate scope, selectivity, activity, stability, and catalytic cycle, it is interesting to outline similarities and differences. In this Review, we intend to delineate which enzymes possess considerable advantages within specific issues. Heterologous production, crystal structure availability, enzyme engineering potential, and substrate promiscuity are essential factors for the applicability of these biocatalysts.

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.

The ipso-substitution of one (or two) hydroxy groups of phloroglucinol with arene nucleophiles (e.g., o-xylene, tetralin, biphenyl) can be achieved easily under Friedel–Crafts-type conditions with or without the use of organic solvents affording a variety of 3,5-dihydroxybiphenyls (57–89% yields). The new method has significant practical advantages compared to classical biaryl-­coupling routes.

The substrate-controlled diastereoselective arylation of chiral aldehydes readily available from carbohydrates is described, using the boron–zinc exchange reaction to generate the transferable aryl groups. The methodology developed was applied to the total synthesis of the styryllactone (+)-7-epi-goniofufurone and analogues thereof.

Photoaffinity tags can be incorporated easily into peptoids and congeners by the Ugi and Passerini multicomponent reactions. Products related to photo-methionine and photo-leucine can be accomplished by diazirine-containing building blocks. The same protocols can be used to synthesize derivatives with benzophenone photo cross-linkers.