Diseño de la peroxigenasa inespecífica de "Agrocybe aegerita" mediante evolución dirigida: expresión funcional en levaduras y síntesis de 1-naftol

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 31-03-2016Han posibilitado este trabajo, tanto proyectos de la Unión Europea (PEROXICATS: FP7-KBBE- 2010-4-26537 e INDOX: FP7-KBBE-2013-7-613549) como Nacionales (Evofacel: BIO2010-19697). Asimismo agradecer la dedicación de COST-Action (CM1303: Systems Biocatalysis

Mapping potential determinants of peroxidative activity in an evolved fungal peroxygenase from agrocybe aegerita

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.This work was supported by the Comunidad de Madrid Synergy CAM Project Y2018/BIO-4738-EVOCHIMERA-CM, the PID2019-106166RB-100-OXYWAVE and the PID2019-106370RB-I00-PZymes grants from the Spanish Ministry of Science and Innovation and the CSIC Project PIE-201580E042. AB-N thanks the PhD Programme in Molecular Biosciences, Doctoral School, Universidad Autónoma de Madrid.Peer ReviewedPostprint (published version

Synthesis of 1‐Naphthol by a Natural Peroxygenase engineered by Directed Evolution

This is the peer reviewed version of the following article, which has been published in final form at 10.1002/cbic.201500493. This article may be used for non-commercial purposes in accordance With Wiley-VCH Terms and Conditions for self-archivingThere 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 H2O2, this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.We thank Paloma Santos Moriano (ICP, CSIC, Spain) for assistance with the HPLC and LC/MS analysis, and Jesper Vind (Novozymes, Denmark) and Angel T. Martinez (CIB, CSIC, Spain) for helpful discussions. This work was supported by the European Commission projects Indox-FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis, and the National Projects Dewry [BIO201343407-R], Cambios [RTC-2014-1777-3] and OXYdesign [CTQ2013-48287-R].Peer ReviewedPostprint (author's final draft

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

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 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Enzymes for consumer products to achieve climate neutrality

29 pags., 4 figs., 3 tabs., 1 graf.Accumulated greenhouse gas emissions are expected to increase from 36.2 Giga-tons (Gt) to 60 Gt over the next three decades. The global surface temperature has increased by¿+¿1.09¿°C since 2001, and might increase by¿+¿2.2¿°C in 2100, +3.6¿°C in 2200 and +4.6¿°C in 2500. These emissions and temperature rises cannot be reduced in their entirety, but they can be lowered by using enzymes. Enzymes are proteins that catalyze biochemical reactions that make life possible since 3.8 billion years ago. Scientists have been able to "domesticate" them in such a way that enzymes, and their engineered variants, are now key players of the circular economy. With a world production of 117 Kilo-tons and a trade of 14.5 Billion-dollars, they have the potential to annually decrease CO2 emissions by 1 to 2.5 Billion-tons (Bt), the carbon demand to synthesise chemicals by 200 Million tons (Mt), the amount of chemicals by 90¿Mt, and the economic losses derived from global warming by 0.5%, while promoting biodiversity and our planet¿s health. Our success to increase these benefits will depend on better integration of enzymatic solutions in different sectors.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. MF also acknowledges Grants PID2020-112758RB-I00, PDC2021-121534-I00, and TED2021-130544B-I00 from the MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”)

Directed evolution of unspecific peroxygenase

Alcalde, Miguel (Ed.).Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase with mono(per)oxygenase activity for the selective oxyfunctionalization of C-H bonds. Fueled by catalytic concentrations of H2O2, which acts as both oxygen donor and as final electron acceptor, this stable, soluble and extracellular enzyme is a potential biocatalyst for dozens of transformations that are of considerable interest in organic synthesis. In this chapter we describe the main attributes of this versatile enzyme, while reflecting on the directed evolution campaigns recently followed in our laboratory that set out to enhance the functional expression of UPO in yeast and improve the activity, as well as approximating its properties to the required industrial standards.We acknowledge the funding and financial support obtained from the European Commission project FP7-KBBE-2013-7-613549-INDOX, the COST-Action CM1303 and the Spanish Government project BIO2013-43407-R-DEWRY.Peer reviewe

Engineering the unspecific peroxygenase, a wide reaction range biocatalyst, by directed evolution

Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014.The unspecific peroxygenase (UPO) is a new type of heme-thiolate enzyme with an exclusive self-sufficient mono(per)oxygenase activity and many attractive applications in organic synthesis amongst other biotechnological uses (1, 2). In this work, the UPO1 gene from the basidiomiycete Agrocybe aegerita was subjected to directed evolution using Saccharomyces cerevisiae as heterologous host. To promote functional expression, several fusions were tested comprising different signal peptides attached to the mature protein. Over 9,000 clones were screened with an ad-hoc dual-colorimetric assay that permitted to assess both peroxidative (ABTS as substrate) and oxygen-transfer activities (NBD as substrate) (3). After five generations of evolution (including random, hybrid and rational approaches such as mutagenic PCR, MORPHING (4) and sitedirected mutagenesis, respectively), 9 mutations were introduced providing a total activity improvement of 3,250-fold without jeopardizing the protein stability. The evolved UPO1 was active and highly stable in the presence of extreme concentrations of organic cosolvents. Mutations at the hydrophobic core of the signal peptide enhanced secretion levels whereas some mutations placed in the neighborhood of the heme accessPeer Reviewe

Mutantes de la peroxigenasa inespecífica con alta actividad monooxigenasa y sus usos

[EN] The invention relates to an unspecific peroxygenase of the Agrocybe aegerita fungus, obtained by means of directed molecular evolution in order to facilitate the functional expression thereof in the active, soluble and stable form. The peroxygenase described in the invention exhibits a significant increase in the functional expression thereof, an improved monooxygenase activity and a reduced peroxydase activity, in relation to the monooxygenase and peroxydase activities exhibited by the wild-type unspecific peroxygenase of A. aegerita. The peroxygenase of the invention can be used in chemical processes, including industrial transformations such as the selective oxyfunctionalisation of carbon-hydrogen bonds of various organic compounds.[ES] La presente invención se refiere a una peroxigenasa inespecífica del hongo Agrocybe aegeritaobtenida mediante evolución molecular dirigida para facilitar su expresión funcional en forma activa, soluble y estable. La peroxigenasa descrita en la presente invención presenta un incremento significativo en su expresión funcional, una actividad monooxigenasa mejorada y una actividad peroxidasa disminuida, respecto a las actividades monooxigenasa y peroxidasa que presenta la peroxigenasa inespecífica silvestre de A. aegerita. La peroxigenasa de la invención es útil en procesos químicos, incluyendo transformaciones industriales como son la oxifuncionalización selectiva de enlaces carbono-hidrógeno de diversos compuestos orgánicos.Consejo Superior de Investigaciones Científicas (España)E Solicitud de patente europe

Mutantes de la peroxigenasa inespecífica con alta actividad monooxigenasa y sus usos

[EN] The invention relates to an unspecific peroxygenase of the Agrocybe aegerita fungus, obtained by means of directed molecular evolution in order to facilitate the functional expression thereof in the active, soluble and stable form. The peroxygenase described in the invention exhibits a significant increase in the functional expression thereof, an improved monooxygenase activity and a reduced peroxydase activity, in relation to the monooxygenase and peroxydase activities exhibited by the wild-type unspecific peroxygenase of A. aegerita. The peroxygenase of the invention can be used in chemical processes, including industrial transformations such as the selective oxyfunctionalisation of carbon-hydrogen bonds of various organic compounds.[ES] La presente invención se refiere a una peroxigenasa inespecífica del hongo Agrocybe aegeritaobtenida mediante evolución molecular dirigida para facilitar su expresión funcional en forma activa, soluble y estable. La peroxigenasa descrita en la presente invención presenta un incremento significativo en su expresión funcional, una actividad monooxigenasa mejorada y una actividad peroxidasa disminuida, respecto a las actividades monooxigenasa y peroxidasa que presenta la peroxigenasa inespecífica silvestre de A. aegerita. La peroxigenasa de la invención es útil en procesos químicos, incluyendo transformaciones industriales como son la oxifuncionalización selectiva de enlaces carbono-hidrógeno de diversos compuestos orgánicos.Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic