Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semi-racionales

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-9-200

Combinatorial Saturation Mutagenesis by in vivo Overlap Extension for the Engineering of Fungal Laccases

Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -CV below 10 %- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with 3-fold better kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and beneficial mutation S510G (TCG/GGG) which can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.This material is based upon work founded by Spanish Ministry of Education and Science (project VEM2004-08559), Comunidad de Madrid/CSIC (project 20058M121) and Ramón y Cajal Research Program. MZ thanks Gobierno Vasco for fellowship.Peer reviewe

Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: The connection between the C-terminal plug and the conserved509VSG511 tripeptide

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The Km O 2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.This research was funded by the Spanish Ministry of Education and Science projects VEM2004-08559, CTQ2005-08925-C02-02/PPQ and CTQ2006-12097/BQU; the EU project NMP2-CT-2006-026456; a CSIC project 200580M121, the Ramón y Cajal Program, and the Swedish Research Council (Vetenskapsårdet, project ärandenummer: 621-2005-3581). MZ was supported by a fellowship from the “Gobierno Vasco” and CV by a fellowship from the FPU program of the “Ministerio de Educación y Ciencia”.Peer Reviewe

Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales

204 páginas, 64 figuras y 29 tablasMimicking the darwinist algorithm of natural selection, through several rounds of random mutagenesis and/or DNA recombination coupled to massive high-throughput screenings, directed molecular evolution allows to tailor enzymes more robust, stable, efficient and in general with many improved features. In this evolutionary scenario, semi-rational analysis - where researches are taking advantage from protein structural information to create and explore libraries constructed by saturation mutagenesis- constitutes also a powerful methodology. Laccases from white-rot fungi are remarkable biocatalysts due to their broad substrate specificity, with potential applications in bioremediation, lignocellulose processing, organic synthesis and more. Most of these transformations must be carried out at high concentrations of organic cosolvents where laccase undergoes unfolding, therefore loosing its catalytic performance. We have evolved the thermostable laccase from the Ascomycete Myceliophthora thermophila to tolerate high concentrations of cosolvents. The genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae, variant R2, was capable to resist a wide array of miscible cosolvents of biotechnological significance at concentrations as high as 50% (v/v). Intrinsic electrochemical laccase features such as the redox potential at the T1 and T2/T3 sites and the geometry and electronic structure of the catalytic coppers were altered during the course of the in vitro evolution experiment. Some mutations located at the surface of protein contributed to the reinforcement of the protein architecture at different key-denaturation regions by establishing new hydrogen bonds and salt bridges. Moreover, the mutations introduced at the C-terminal extension affected the protein folding at the post-translational maturation steps. Additionally, we have developed a new methodology named in vivo overlap extension (IVOE) which is based on the high homologous recombination frequency of Saccharomyces cerevisiae. This methodology provides a simple manner to build combinatorial saturation mutagenesis libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. Several positions that seem to be related with the redox potential at the T1 site were targeted for combinatorial saturation mutagenesis. After exploring over 170000 clones, the best variant revealed a direct relationship between the highly conserved tripeptide 509VSG511, located in the vicinity of the T1 site, and the C-terminal plug. The Km O2 value of the mutant was increased 1.5-fold and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, thus improving catalytic efficiencies about 3- and 8-fold towards nonphenolic and phenolic substrates, respectively. Although the copper geometry at the T1 site was perturbed upon mutation, paradoxically the laccase redox potential was not significantly altered. Taking together, the present study suggests that 509VSG511 tripeptide may play a hithertounrecognized role in regulating the traffic of O2 to the T2/T3 copper cluster, in combination with the C-terminal plug, which is dependent on the binding of the reducing substrate at the copper T1.Peer reviewe

Combinatorial Saturation Mutagenesis by in vivo Overlap Extension for the Engineering of Fungal Laccases

Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -CV below 10 %- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with 3-fold better kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and beneficial mutation S510G (TCG/GGG) which can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.This material is based upon work founded by Spanish Ministry of Education and Science (project VEM2004-08559), Comunidad de Madrid/CSIC (project 20058M121) and Ramón y Cajal Research Program. MZ thanks Gobierno Vasco for fellowship.Peer reviewe

Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

11 páginas, 5 figuras, 4 tablas -- PAGS nros. 250-260The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with ∼3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccasesMZ thanks Gobierno Vasco for a fellowship.Peer reviewe

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.This work was supported by grants from ViaLactiaBiosciences Ltd. (New Zealand), the BMBF BiotechGenoMik Programme (to P. N. G. and O. V. G.), European Community Project MERG-CT-2004-505242 “BIOMELI,” Spanish Ministry of Education and Science (VEM2004-08559) and Comunidad de Madrid Project (GR/AMB/0690/2004), Gobierno Vasco (to M. Z.), a European Community Marie Curie fellowship, a Spanish Ministerio de Ciencia y Tecnología-Ramón y Cajal contract (to M. F.), and Madrid Autonomic Community and European Social Funds (to M. P.).Peer reviewe