Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae.

For more than thirty years, biotechnology has borne witness to the power of directed evolution in designing molecules of industrial relevance. While scientists all over the world discuss the future of molecular evolution, dozens of laboratory-designed products are being released with improved characteristics in terms of turnover rates, substrate scope, catalytic promiscuity or stability. In this review we aim to present the most recent advances in this fascinating research field that are allowing us to surpass the limits of nature and apply newly gained attributes to a range of applications, from gene therapy to novel green processes. The use of directed evolution in non-natural environments, the generation of catalytic promiscuity for non-natural reactions, the insertion of unnatural amino acids into proteins or the creation of unnatural DNA, is described comprehensively, together with the potential applications in bioremediation, biomedicine and in the generation of new bionanomaterials. These successful case studies show us that the limits of directed evolution will be defined by our own imagination, and in some cases, stretching beyond that.

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.

Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values. The full ensemble of segments (43% of UPO sequence) was subjected to focused evolution by the Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) method in Saccharomyces cerevisiae. Five independent mutant libraries were screened in terms of P:p ratio and thermostability. We identified several variants that harbored substitutions at positions 120 and 320 with a strong enhancement in the P:p ratio albeit at the cost of stability. The most thermostable mutant of this process (S226G with an increased T50 of 2°C) was subjected to further combinatorial saturation mutagenesis on Thr120 and Thr320 yielding a collection of variants with modified P:p ratio and recovered stability. Our results seem to indicate the coexistence of several oxidation sites for peroxidative and peroxygenative activities in UPO.

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere’s carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.