RESUMO
The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4â g L-1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4â g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.
Assuntos
Ácidos Graxos , Oxigenases de Função Mista , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Ácidos Graxos/química , Oxirredução , HidroxilaçãoRESUMO
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.
Assuntos
Agrocybe , Evolução Molecular Direcionada , Proteínas Fúngicas , Oxigenases de Função Mista , Mutação de Sentido Incorreto , Solventes/química , Acetona/química , Acetonitrilas/química , Agrocybe/enzimologia , Agrocybe/genética , Dimetil Sulfóxido/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Metanol/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genéticaRESUMO
Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.
Assuntos
Evolução Molecular Direcionada , Enzimas Imobilizadas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Animais , Bovinos , Fluoresceína-5-Isotiocianato/metabolismo , Mutação/genética , Engenharia de Proteínas , Saccharomyces cerevisiaeRESUMO
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae, so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivoIMPORTANCE 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.
Assuntos
Deriva Genética , Oxigenases de Função Mista/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Embaralhamento de DNA , Estabilidade Enzimática , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Mutação , Filogenia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2 O2 , this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.
Assuntos
Agrocybe/enzimologia , Evolução Molecular Direcionada , Oxigenases de Função Mista/metabolismo , Naftalenos/metabolismo , Naftóis/metabolismo , Engenharia de Proteínas , Agrocybe/genética , Agrocybe/metabolismo , Oxigenases de Função Mista/genética , Modelos Moleculares , Mutação PuntualRESUMO
Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.
Assuntos
Agrocybe/enzimologia , Evolução Molecular Direcionada , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/enzimologia , Agrocybe/genética , Colorimetria/métodos , Estabilidade Enzimática , Perfilação da Expressão Gênica , Testes Genéticos , Cinética , Oxigenases de Função Mista/química , Sinais Direcionadores de Proteínas/genética , Saccharomyces cerevisiae/genéticaRESUMO
The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).
Assuntos
Esterases , Lipase , Esterases/metabolismo , Lipase/metabolismo , Fosfolipases , Ensaios de Triagem em Larga Escala/métodosRESUMO
Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.
RESUMO
[This corrects the article DOI: 10.3389/fbioe.2021.741282.].
RESUMO
The most famous yeast of all times, Saccharomyces cerevisiae, has been used by humankind for at least 8000 years, to produce bread, beer and wine, even without knowing about its existence. Only in the last century we have been fully aware of the amazing power of this yeast not only for ancient uses but also for biotechnology purposes. In the last decades, wine culture is becoming more and more demanding all over the world. By applying a powerful biotechnological tool as genetic engineering in S. cerevisiae, new horizons appear to develop fresh, improved or modified wine characteristics, properties, flavors, fragrances or production processes, to fulfill an increasingly sophisticated market that moves around 31.4 billion per year.
RESUMO
Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C-H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.
Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Agrocybe/enzimologia , Domínio Catalítico/genética , Cristalografia por Raios X , Evolução Molecular Direcionada , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligantes , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Pichia/genética , Ligação Proteica , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato/genéticaRESUMO
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.
Assuntos
Agrocybe , Evolução Molecular Direcionada , Proteínas Fúngicas , Oxigenases de Função Mista , Agrocybe/enzimologia , Agrocybe/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
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.
Assuntos
Bioengenharia , Materiais Biomiméticos , Evolução Molecular Direcionada , Nanoestruturas , Biologia Sintética , Biotecnologia , DNA/química , DNA/genética , DNA/metabolismoRESUMO
Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (â¼8mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.
Assuntos
Agrocybe/enzimologia , Evolução Molecular Direcionada/métodos , Proteínas Fúngicas/biossíntese , Oxigenases de Função Mista/biossíntese , Pichia/metabolismo , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Agrocybe/genética , Oxirredutases do Álcool/genética , Fermentação , Proteínas Fúngicas/genética , Genes Fúngicos , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.