Directed and computational evolution of fungal laccases: redox potential enhancement and development of a family of thermostable chimeras

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Universidad Autónoma de Madrid

Programa de Doctorado en Biociencias Moleculares

DIRECTED AND COMPUTATIONAL EVOLUTION OF FUNGAL LACCASES:

REDOX POTENTIAL ENHANCEMENT AND DEVELOPMENT OF A FAMILY OF THERMOSTABLE CHIMERAS

IVAN MATELJAK

TESIS DOCTORAL

Madrid, 2018

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Departamento de Biología Molecular Facultad de Ciencias

Universidad Autónoma de Madrid

DIRECTED AND COMPUTATIONAL EVOLUTION OF FUNGAL LACCASES:

REDOX POTENTIAL ENHANCEMENT AND DEVELOPMENT OF A FAMILY OF THERMOSTABLE CHIMERAS

Institute of Catalysis and Petrochemistry (ICP) Spanish Council for Scientific Research (CSIC)

Ivan Mateljak

Licenciado en Biología Molecular

Director: Dr. Miguel Alcalde Galeote

Tutor: Dr. Francisco Plou Gasca

TESIS DOCTORAL Madrid, 2018

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ACKNOWLEDGEMENTS

At the end of this journey, I would like to express my very great appreciation to all the people who helped and supported me throughout these years in Madrid.

First and foremost, I must give enormous thanks to my supervisor Dr. Miguel Alcalde, first of all for selecting me to join his group at ICP after “technical problem” interview and introducing me to a wonderful world of directed evolution. I am truly grateful for his patient guidance, dedication, enthusiastic encouragement, willingness to give his time and ever-present support during these four and a half years.

I would like to express my deep gratitude to my tutor Dr. Francisco Plou, for his kindness, continuous disposition whenever I needed something and his numerous advices.

Special thanks to Dr. Antonio Ballesteros for his wisdom, high spirit and cordiality.

This Thesis was carried out within the FP7 project BIOENERGY “Biofuels cells: from fundamentals to applications of bioelectrochemisty (FP7-PEOPLE-2013-ITN-607793)”. The development of this Thesis also counted on the support of the Spanish national grants LIGNOLUTION “Directed and computacional evolution of ligninases BIO2016-79106-R” and DEWRY “Directed evolution of modern and ancestral ligninolytic oxidoreductases for the design of a white-rot yeast BIO2013-43407-R”. Particularly, it is worth mentioning that in the BIOENERGY project I had the opportunity to enjoy of an ITN Marie Curie fellowship together with unique multidisciplinary and friendly environment. I truly thank all the partners involved in:

Ruhr-University Bochum, Malmö University, University of Limerick, National University of Ireland (Galway), Lund University, University of Natural Resources and Life Sciences (Vienna), University of Southampton, CNRS Bordeaux, University of Warsaw, Dropsens, Nanoflex Limited and Nanoinnova Technologies. I want to thank all the partners for the opportunity to learn invaluable scientific and personal lessons. Special thanks to Prof. Wolfgang Schuhmann from Ruhr-Universität Bochum for coordinating, leading and guiding this wonderful project to a happy and fruitful end. Also, many thanks to Sabine Seisel for her constant support in all the administrative issues that arose along the course of the project. I am grateful to all PhD students and PostDocs with whom I have had the pleasure to work in BIOENERGY: Chiara, Asier, Elena, Su, Magdalena, Galina, Marta, Francesca, Sabine, Valentina, Nikola, Till, Xin-Xin, Ali, Kanso,

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Bearing in mind that the development of this Thesis was in a multidisciplinary ambience, many different techniques and methods were applied under the careful supervision of several colleagues. Firstly, I am particularly grateful to Dr. Roland Ludwig and Dr. Roman Kittl from University of Natural Resources and Life Sciences in Vienna for giving me opportunity to spend three months in their laboratory where I learned to work with bioreactors. During that period, the assistance provided by Florian Csarman was greatly appreciated. Also, I need to thank Dr. Rafael Ferritto from Nanoinnova Technologies for enabling my short stay in his company where I learned new concepts and techniques. I would also like to thank Dr. Donal Leech from University of Galway for his advices in designing screening assays. Thanks to everyone in Dr. Antonio Lopez de Lacey and Marcos Pita group, from ICP-CSIC, for answering many questions about electrochemistry. My gratitude goes to Dr. Victor Guallar and his group in Barcelona Supercomputing Center for providing constructive suggestions and results, facilitating my work.

I am grateful to Dr. Sergey Shleev and his student Olga Aleksejeva from Malmö University who helped us with the important electrochemical studies. Also, thanks to Thierry Tron from Aix Marseille University for his advices and for providing Lac3 gene. Last but not least, special thanks to Dr. Frances H. Arnold, Austin Rice and Kevin Young from California Institute of Technology (CALTECH) for their invaluable assistance in the SCHEMA project.

I must give special gratitude to my lab-mates in directed enzyme evolution group, without them everything would be much harder. To David who was my first supervisor, who showed me and taught me many things, especially when I came to the lab. To Patri, for her kindness and help whenever I needed. To “birds”, Berni and Xavi, for all the laughter that we shared these years, for all their friendship and support. To Pati, for being my conference companion and always helpful friend. To Javi and Isa for their help and great time spent together, particularly on Rayo matches! To Diana, for her advices about laccases and directed evolution. To many kind people who passed through lab during my time here: Elvin, Sofia, Leire, Katarina, Jia, Berndjan, Andres, Mehdi, Joaquin, Mar and many others.

I am very grateful to all members of Fancicso Plou’s group, our friends from second floor:

Paloma, Noa, Fadia, Joselu, Lucia, Barbara and David for their kindness, help and assistance through all these years.

I would like to thank other ICP members that shared time with me. All the nice people I have met:

Lara, Cristina, Alejandro, Janaina, Maria, Rita, Javi, Sandro, Monica, Rafa, Cristina and many others.

Among them I am particularly grateful to Lara for her great friendship and support, her immense help in paperwork for this Thesis and for driving me every day to work. Together with Chiara, for trying new food in Madrid and spending fun time together.

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I wish to thank my friends from Croatia that I met in Madrid: Barbara, David, Ivan, Lucija, Dora, Iva and many others for their friendship and for bringing a bit of my country closer to me.

I am especially grateful to my childhood friends from Croatia: Nikola, Denis, Zvone, Bustro, Velo, Koko and Ante for their and constant support. Although we were far away, we always found a way to spent great time together either in Spain or Croatia.

I would like to express my deep gratitude to my friend Marija, who provided unending inspiration and care before and during these years.

Nobody has been more important to me in this journey than my family. I would like to thank my parents, whose love and guidance are with me in whatever I pursue. My sisters and my grandma for always believing in me and encouraging me even being far away. Thank you for everything!

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SUMMARY

Fungal laccases are multicopper oxidases with a broad substrate specificity that is highly dependent on their redox potential of the T1Cu site (ET1). High-redox potential laccases (HRPLs) secreted by basidiomycete white rot fungi are particularly relevant in a biotechnological context given their capacity to oxidize compounds with a higher redox potential that cannot be transformed by their medium- and low-redox potential counterparts. In the first section of this Thesis, we combined computational design with directed evolution in order to tailor a HRPL variant with improved ET1 and activity towards high-redox potential mediators, as well as enhanced stability. Laccase mutant libraries were screened in vitro using high-redox potential mediators with different chemical nature, while computer-aided evolution experiments were run in parallel to guide bench-top mutagenesis. Through this strategy, the ET1 of the evolved HRPL increased from 740 mV to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic parameters for high-redox potential mediators were markedly enhanced, which were then successfully tested within laccase mediator systems (LMS). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects and they were rationalized at the atomic level.

Due to its complex structural organization, the generation of chimeric laccases with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. In the second section of this Thesis, using SCHEMA-RASPP structure-guided recombination in vivo, we obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with 70 % protein sequence identity and varied redox potential. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computational SCHEMA blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated and it was screened at high temperature. The collection of chimeras showed considerable sequence diversity, on average varying from their closest parent homolog in 46 amino acids. The most thermostable variant increased its half-life of thermal inactivation at 70°C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acid pH.

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RESUMEN

Las lacasas fúngicas son oxidasas multicobre con una amplia especificidad de sustrato que es altamente dependiente de su potencial de oxidorreducción en el sitio del cobre T1 (ET1).

Las lacasas de alto potencial redox (HRPLs) secretadas por los hongos basidiomicetos de podredumbre blanca son particularmente relevantes en diversos contextos biotecnológicos, dada su capacidad para oxidar compuestos con mayores potenciales redox que no pueden ser transformados por sus lacasas equivalentes con potenciales redox medios o bajos. En la primera sección de esta Tesis Doctoral, se han combinado herramientas de evolución dirigida y diseño computacional para obtener una variante HRPL con mejoras en su ET1 y actividad frente a mediadores de alto potencial redox, así como estabilidad aumentada. Las librerías de lacasas mutantes fueron exploradas in vitro empleando mediadores de alto potencial redox de diferente naturaleza química mientras que se corrieron en paralelo simulaciones de evolución in silico/computacional para guiar la mutagénesis experimental. A través de esta estrategia dual, el ET1 de la HRPL evolucionada incremento de 740 mV to 790 mV, con la correspondiente mejora asociada a su estabilidad termal y frente a pHs ácidos. Los valores cinéticos para mediadores de alto potencial redox se vieron notablemente incrementados, y éstos fueron empleados exitosamente dentro de sistemas lacasa mediador (LMS). Las dos substituciones hidrofóbicas en los alrededores del sitio del cobre T1 son responsables de tales efectos, y se racionalizaron a nivel átomico mediante simulaciones computacionales.

Debido a su compleja organización estructural, la generación de lacasas quiméricas con alta diversidad de secuencia a partir de diferentes ortólogos es difícil de conseguir sin comprometer la función de la proteína. En la segunda sección de esta Tesis Doctoral, a partir de tres lacasas fúngicas con un 70% de identidad de secuencia y empleando recombinación structural-guiada mediante SCHEMA-RASSP in vivo, se ha obtenido una familia diversa en lacasas quiméricas funcionales que muestran incrementos notables de termostabilidad. Ayudados por la elevada frecuencia de recombinación homóloga de DNA de la levadura Saccharomyces cerevisiae, los bloques computacionales de SCHEMA se ensamblaron y clonaron en un único paso de transformación. Como resultado de este ensamblaje in vivo, se generó con facilidad una librería enriquecida en lacasas quiméricas que fue explorada a altas temperaturas. La colección de quimeras mostró una considerable diversidad de secuencia, con un promedio mutacional de 46 amino ácidos frente al parental más próximo. La variante más termoestable incrementó su vida

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ACRONYMS

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) Ag/AgCl Silver/silver chloride electrode

bp Base pairs

BSA Bovine serum albumin CFU Colony forming unit CPU Central processing unit

CSM Combinatorial saturation mutagenesis D1, D2, D3 Cupredoxin domains 1, 2, 3

DMP 2,6-Dimethoxyphenol DMSO Dimethyl sulfoxide dNTPs Deoxyribonucleotides

<E> SCHEMA energy

E Redox potential

ET1 Redox potential at T1 site epPCR Error-prone PCR

ET Electron transfer GE Graphite electrode

GUA Guaiacol

HAT Hydrogen-atom transfer HBT 1-hydroxybenzotriazole HPI N-hydroxyphthalimide HRPLs High redox potential laccases HTS High throughput screening

kb kilobase

kcat Catalytic constant kcat/Km Catalytic efficiency Km Michaelis-Menten constant LMS Laccase mediator system

<m> Average number of mutations in library relative to the closest parent MSA Multiple sequence alignment

MORPHING Mutagenic Organized Recombination Process by Homologous IN vivo Grouping MtL Myceliopthora thermophila laccase

NHA N-hydroxyacetanilide

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NHE Normal hydrogen electrode OCM Octacyanomolybdate OCP Open circuit potential OD600 Optical density at 600 nm

PAHs Polycyclic aromatic hydrocarbons

PAOX1 Alcohol oxidase I promoter

PcL Pycnoporus cinnabarinus laccase PDB Protein data bank

PELE Protein Energy Landscape Exploration PM1L PM1 laccase

QM/MM Quantum mechanics/Molecular mechanics RASPP Recombination as Shortest Path Problem RB5 Reactive black 5

RMSD Root-mean-square deviation

SA Sinapic acid

SEM Selective expression medium SM Saturation mutagenesis

t1/2 Time required by the enzyme to lose 50% of its initial activity upon incubation at a given temperature

T1Cu Type 1 copper

VLA Violuric acid

α^native Native α factor prepro-leader

α^PcL Evolved α factor prepro-leader for PcL secretion

α^PM1L Evolved α factor prepro-leader for PM1L secretion

Ɛ Extinction molar coefficient

 Overpotential

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1 INTRODUCTIO N

1 INTRODUCTION

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INTRODUCTION

1.1 Laccases

1.1.1 General aspects and distribution in nature

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) catalyze the oxidation of a wide array of substrates using molecular oxygen as final electron acceptor. The catalytic structure of this enzyme is organized in two centers composed of 4 copper atoms: One copper atom at the T1 site, where the substrate is oxidized and the trinuclear cluster, consisting of one T2 and two T3 coppers, where reduction of molecular oxygen takes place. Laccases belong to the family of multi-copper oxidases together with ascorbate oxidase from plants, human ceruloplasmin, bacterial nitrite reductase, fungal ferroxidase and bilirubin oxidase from fungi and insects (Baldrian 2006; Hoegger et al. 2006). These enzymes are extensively distributed in fungi (mostly white rot) (Brijwani, Rigdon, and Vadlani 2010) bacteria (Santhanam et al. 2011) and higher plants (Mayer and Staples 2002) performing a variety of biological functions depending on their origin and life stage of the producing organism. They have been also reported in sponges (Li et al. 2015) and lichens (Laufer et al. 2006), while laccase-like activity has been detected in oysters (Luna-Acosta et al. 2010) insect cuticles (Lang, Kanost, and Gorman 2012) and metagenome libraries of bovine rumen (Beloqui et al. 2006). In fungi, laccases are implicated in lignin degradation, stress defense, morphogenesis, and fungal plant-pathogen/host interactions (Alcalde 2007). Function of bacterial laccases is connected with morphogenesis, pigmentation, toxin oxidation and protection against oxidizing agents and UV light (Singh et al. 2011), while plant laccases are acting on lignin polymerization and wound response (Mayer and Staples 2002). In the rest of the organisms with identified laccase activity, the role of this enzyme is still unknown.

1.1.2 Structural and functional characteristics

Laccase from Coprinus cinereus was the first laccase structure solved albeit missing the T2Cu (Ducros et al. 1998). Currently, numerous laccase structures from different organisms are available including those from Bacillus subtilis, Melanocarpus albomyces, Coriolopsis gallica, Pycnoporus cinnabarinus, Trametes versicolor, Trametes trogii, Rigidoporus lignosus, Cerrena maxima, Coriolus zonatus and Lentinus tigrinus (Mot and Silaghi-Dumitrescu 2012). Laccases are mostly extracellular monomeric glycoproteins with molecular masses between 50 and 130 kDa and a glycosylation degree of 45 % in plant laccases and 10-20 % in fungal laccases, respectively. Its overall structure is organized in 3 cupredoxin domains, Figure 1.1. The main structural feature of each domain is β barrel, motif observed in single copper proteins such as azurin, rusticyanin, plastocyanin being ordinary in all multicopper oxidases, which suggests the

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INTRODUCTION

promoting stability (Mot and Silaghi-Dumitrescu 2012). The structure is further stabilized with two or three disulfide bridges between domains: in the case of basidiomycete PM1 laccase, which is departure point of this Thesis, two disulfide bridges are included (the first connecting domains 1 and 3 and the second bridging domains 1 and 2, respectively). As mentioned above, four copper atoms are within the laccase structure, which can be classified into three types based on their unique spectroscopic features.

 Type 1 copper (T1Cu) or blue paramagnetic copper in oxidized resting state site exhibits intense absorption at 610 nm (ε610 5,000 M^-1 cm^-1) due to a highly covalent Cu-S bond.

T1Cu is further coordinated by nitrogen atoms in position δ1 (Nδ1) of two histidine residues resulting in three coordinated trigonal planar geometry.

 Type 2 copper (T2Cu) is a paramagnetic non-blue copper atom which shows no significant absorbance feature except normal EPR spectrum parameters. It is coordinated by two nitrogen atoms in position ε2 (Nε2) of two histidine residues and one oxygen atom from water.

 Binuclear Type 3 copper (T3Cu) sites are anti-ferromagnetically coupled through a hydroxyl bridge in the resting oxidized state resulting in total absence of EPR signal. This hydroxyl bridge is also responsible for a shoulder in the UV spectrum at 330nm. In total, six histidine residues and one water molecule are coordinating the T3 pair, resulting that each copper is tetracoordinated in a trigonal bipyramidal geometry. Five histidine residues are bound to coppers by Nε2 atoms and remaining one by Nδ1 atom.

Electrons captured from substrate at T1Cu site are transferred via a His-Cys-His tripeptide to the trinuclear cluster (situated approximately 12 Å from T1Cu) where oxygen is reduced. Both copper coordinating residues and the His-Cys-His tripeptide are highly conserved. Laccase PM1 structure is also characterized by two solvent channels which provide access to the trinuclear copper cluster.

The first channel points towards the two T3 copper ions on one side of the T2/T3 cluster allowing the molecular oxygen to enter and bind to it. On the other side of the cluster, the second channel points towards the T2Cu allowing water -produced upon O2 reduction- to flow to the bulk solvent (Matera et al. 2008). Although being intensively investigated in last decades, the electron transfer pathway and the mechanism of oxygen reduction in laccases are still not fully elucidated.

Nevertheless, certain aspects have been already established.

1) One-electron oxidation of the reducing substrate occurs at the T1Cu site. Free cationic radical is generated by laccase which can undergo further non-enzymatic polymerization or hydration.

2) Water production from oxygen at the T2/T3 site requires four electrons implying four reducing substrates molecules to be oxidized. Consequently, laccase is operating as a sort of “battery”,

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INTRODUCTION

accumulating electrons from individual oxidation reactions at the T1Cu site for the reduction of molecular oxygen to water.

3) Four electrons are transferred through the His-Cys-His tripeptide to the trinuclear copper cluster where one molecule of oxygen is bound and reduced to two molecules of water.

Spectroscopic, kinetic, and computational data have shown that oxygen reduction is occurring via a peroxide intermediate.

Figure 1.1 Overall laccase structure and intramolecular electron transfer pathway. Domain 1, red.

Domain 2, green. Domain 3, purple. Copper atoms are represented as blue spheres. His and Cys residues are depicted as sticks. His-Cys-His tripeptide is highlighted in cyan (PDB code 2HRG).

Laccases have a very broad substrate spectrum, although they act preferably on phenolic compounds. The range of substrates accepted by laccases is varying from one laccase to another and that makes difficult to establish with accuracy the definition of laccase activity. Generally, laccases are capable to oxidase o- and p-diphenols, methoxy-substituted phenols, aminophenols, polyphenols, benzenethiols, polyamines, hydroxyindols and some aryl diamines.

Inorganic/organic metal complexes are also laccase substrates (e.g. [W(CN)8]^4‐, [Mo(CN)8]^4‐, [Fe(EDTA)]^2‐). Furthermore, laccase can act on redox mediators such as (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid , (ABTS) or 1-hydroxybenzotriazole , (HBT) (Morozova et al. 2007). Laccase substrate spectrum is highly dependent on the redox potential of T1Cu site, which constitutes the upper limit to their oxidation capacity and will be discussed in the following

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INTRODUCTION

results in a blockage of the intramolecular electron transfer with the consequent inhibition of activity. This is also the reason of enzyme inactivation at basic pH. Other laccase inhibitors are fatty acids, sulfhydryl reagents, metal ions (e.g. Hg²⁺), kojic acid, desferal, hydroxyglycine and cationic quaternary ammonium detergents, in reactions that may involve amino acid residue modifications, conformational changes or copper chelation (Call and Mücke 1997; Gianfreda, Xu, and Bollag 1999).

1.1.3 Redox potential at T1Cu site

The redox potential (E) can be defined as the electrochemical driving force required to capture electron(s) from a reducing substrate. In general terms, the higher the E the stronger the affinity for electrons (Rodgers et al. 2010). As such, redox potential of the T1Cu site plays a crucial role in the overall performance of these enzymes. Indeed, it is well known that the electron subtraction from the substrate to the T1Cu is the rate-limiting step in laccase catalysis and the difference between the redox potentialof the substrate and the T1Cu is the driving force of reaction (Jones and Solomon 2015). Xu and coworkers showed that higher difference of redox potential between T1Cu site of laccase and reducing substrate is correlated with a higher catalytic efficiency (Xu 1996; Xu et al. 1996; Xu et al. 1999) The importance of these findings is related to the laccase substrate scope indicating the crucial role of redox potential at T1Cu site and the ability of laccase to expand substrate spectrum to compounds with higher redox potential.

Laccases have been classified as low-, medium- and high-redox potential based on equilibrium potentiometric titrations of the T1Cu site which is ranging from 370 to 790 mV vs.

NHE (Normal Hydrogen Electrode), Table 1.1 (Shleev et al. 2005). High-redox potential laccases (HRPLs) are found almost exclusively in basidiomycete fungi, while bacterial and plant laccases usually exhibit lower redox potential. From biotechnological point of view, HRPLs are relevant by showing strong catalytic capacity and oxidizing molecules with higher redox potentialthat cannot be transformed by their medium- and low-redox potential counterparts (Kunamneni et al.

2008; Shleev et al. 2005). In addition, a high ET1 offers an attractive set of advantages for engineering biodevices (biosensors and high-redox potential enzymatic cathodes of biofuel cells), allowing the laccase to accept electrons directly from the cathode, to generate higher current densities, and to lower the overpotential for O2 reduction (Shleev et al. 2005; Falk, Blum, and Shleev 2012).

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INTRODUCTION

Table 1.1 Redox potential at the T1Cu site (E_T1) of laccases from different organisms and the corresponding sequence alignments.

Laccase source Organism ET1, mV (vs. NHE)

Sequence alignment Reference HIGH REDOX POTENTIAL

Trametes ochracea Basidiomycete +790 ± 10 ⁴⁵²H C H I D F H L E A G F⁴⁶³ (Shleev et al. 2004) Trametes trogii Basidiomycete +790 ⁴⁴⁹H C H I D F H L E A G F⁴⁶⁰ (Garzillo et al. 2001) Trametes versicolor Basidiomycete +785 ⁴⁵²H C H I D F H L E A G F⁴⁶³ (Reinhammar 1972) Botrytis cinerea Ascomycete +780 ⁵⁰⁸H C H I A W H A S E G L⁵¹⁹ (Li, Xu, and Eriksson

1999)

Coriolopsis fulvocinerea Basidiomycete +780 ± 10 n.d. (Shleev et al. 2004) Trametes hirsuta Basidiomycete +780 ± 10 ⁴⁵²H C H I D F H L D A G F⁴⁶³ (Shleev et al. 2004) Trametes villosa Basidiomycete +780 ⁴⁵²H C H I D F H L E A G F⁴⁶³ (Li, Xu, and Eriksson

1999)

Cerrena maxima Basidiomycete +750 ± 5 ⁴⁵²H C H I D F H L E G G F⁴⁶³ (Shleev et al. 2004) Pycnoporus cinnabarinus Basidiomycete +750 ⁴⁵⁰H C H I D F H L E A G F⁴⁶¹ (Li, Xu, and Eriksson

1999)

Trametes pubescens (LAC1) Basidiomycete +746 ± 5 ⁴⁵⁷H C H I D F H L E A G F⁴⁶⁸ (Shleev et al. 2007)

RL5 Metagenomic +745 n.d. (Beloqui et al. 2006)

Pleurotus ostreatus (POXC) Basidiomycete +740 ⁴⁶⁰H C H I D W H L E I G L⁴⁷¹ (Garzillo et al. 2001) Trametes pubescens (LAC2) Basidiomycete +738 ± 5 ⁴⁵²H C H I D F H L E A G F⁴⁶³ (Shleev et al. 2007) Trametes sp. C30 (LAC1) Basidiomycete +730 ⁴⁴⁹H C H I D F H L E A G F⁴⁶⁰ (Klonowska et al.

2002)

Botrytis aclada Ascomycete +720 ⁵²⁵H C H I A W H A S E G L⁵³⁶ (Osipov et al. 2014) MEDIUM REDOX POTENTIAL

Rhizoctonia solani Basidiomycete +710 ⁴⁵⁹H C H I D W H L E A G L⁴⁷⁰ (Xu et al. 1998) Rigidoporus lignosus Basidiomycete +700 ⁴⁷²H C H I D W H L E A G L⁴⁸³ (Bonomo et al. 1998) Trichoderma harzianum

WL1

Ascomycete +692 n.d. (Sadhasivam et al.

2008) Pleurotus ostreatus

(POXA1b)

Basidiomycete +650 ⁴⁵⁰H C H I D W H L D L G F⁴⁶¹ (Garzillo et al. 2001)

Trametes sp. C30 (LAC2) Basidiomycete +560 ⁴⁵²H C H I D F H L E A G F⁴⁶³ (Klonowska et al.

2002)

Coprinus cinereus Basidiomycete +550 ⁴⁵¹H C H I E F H L M N G L⁴⁶² (Schneider et al. 1999) Trichophyton rubrum LKY-7 Ascomycete +540 ⁵³⁰H C H I A W H S S Q G L⁵⁴¹ (Jung, Xu, and Li

2002)

CueO from Escherichia coli Bacteria +500^a ⁴⁷¹H C H L L E H E D T G M⁴⁸² (Miura et al. 2009) Scytalidium thermophilum Ascomycete +510 ⁵⁰⁶H C H I A W H V S G G L⁵¹⁷ (Xu et al. 1998) Melanocarpus albomyces Ascomycete +470 ⁵⁰²H C H I A W H V S G G L⁵¹³ (Andberg et al. 2009) Myceliophthora thermophila Ascomycete +470 ⁵⁰²H C H I A W H V S G G L⁵¹³ (Xu et al. 1998) LOW REDOX POTENTIAL

CotA from Bacillus subtilis Bacteria +455 ⁴⁹¹H C H I L E H E D Y D M⁵⁰² (Melo et al. 2007) CueO from Escherichia coli Bacteria +440^b ⁴⁷¹H C H L L E H E D T G M⁴⁸² (Miura et al. 2009) Rhus vernicifera Plant +434 ⁴⁹⁵H C H F E R H T T E G M⁵⁰⁶ (Reinhammar 1972) SLAC from Streptomyces

coelicolor

Bacteria +430 ²⁸⁷H C H V Q S H S D M G M²⁹⁸ (Gallaway et al. 2008)

McoP from Pyrobaculum aerophilum

Bacteria +398 ⁴³⁰H C H N L E H E D G G M⁴⁴¹ (Fernandes et al. 2010)

Ssl1 from Streptomyces sviceus

Bacteria +375 ± 8 ²⁸⁴H C H V Q S H S D M G M²⁹⁵ (Gunne et al. 2014)

The isoenzyme is in parentheses. The T3Cu ligands are in italics, T1Cu ligands are underlined and the T1Cu axial ligand is in bold. ^aET1 determined at pH 5.0. ^bET1 determined at pH 7.0.

Despite having very similar T1Cu coordination geometry, redox potentialcan vary significantly among laccases (Shleev et al. 2004; Hong et al. 2011). Two histidine residues and a cysteine,

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INTRODUCTION

in bacterial and plant laccases) whereas in fungal laccases, leucine or phenylalanine are placed on such position (in non-coordinating interaction) contributing, to some extent, to their higher redox potential (Durao et al. 2006). Indeed, multiple sequence alignment (MSA) and spectroelectrochemical characterizations address laccases with the highest redox potential harboring a phenylalanine residue as axial ligand of T1Cu, Table 1.1. (Tadesse et al. 2008).

Several site-directed mutagenesis studies were performed trying to elucidate the role of the axial ligand on ET1. The mutations F463M in Trametes villosa laccase and L499M in Botrytis aclada laccase resulted in a decrease of redox potential for approximately 100mV (Xu et al. 1999; Osipov et al. 2014). Work of Durao and coworkers showed similar results mutating methionine to leucine in bacterial CotA laccase which increased ET1 by 100 mV but at the expense of decreasing both the thermodynamic stability and the activity for all substrate tested (Durao et al. 2006). On the other hand, mutating the axial ligand from leucine to phenylalanine in Myceliopthora thermophila laccase (ET1 = 470 mV vs. NHE) did not led to any significant changes in enzymatic properties including the redox potential which indicates that the nature of axial ligand is not the only factor that affect ET1 (Xu et al. 1998). Indeed, it has been proposed that many other parameters could influence the redox potentialin blue copper proteins such as the hydrophobicity in T1 site environment, the solvent accessibility, the distances between the T1Cu, the coordinating histidines and cysteine, the hydrogen bonding to the S(Cys), and stacking and electrostatic interactions in the protein backbone (Palmer et al. 1999; Marshall et al. 2009; Hong et al. 2011;

Cambria et al. 2012; Hadt et al. 2012). It was demonstrated in the case of azurin, a single copper cupredoxin, fine redox potential tuning over a wide range via hydrophobicity and hydrogen- bonding, but in laccases, proteins with more complex redox centers and electron transfer, we lack those kinds of studies (Marshall et al. 2009). How to modify the physico-chemical properties affecting ET1 of laccase and which other residues or protein segments could be involved in its alteration remains a challenge and that question will be investigated in this Thesis.

1.1.4 Laccase mediator system

Laccases redox potential does not exceed 800 mV which results in the inability of the enzyme to transform directly polycyclic aromatic hydrocarbons (PAHs), many recalcitrant dyes and nonphenolic lignin structures with high redox potential, such as 1,2-dimethoxybenzene, veratryl alcohol and others. (Alcalde, Bulter, and Arnold 2002; Galli and Gentili 2004; Morozova et al. 2007). However, laccases can enhance their substrate range by employing redox mediators which behave as diffusible electron shuttles between the enzyme and the substrates, enabling their oxidation while surpassing steric hindrance and differences in redox potentials. Therefore, industrial applicability of laccase may be expanded by the use of laccase-mediator system (LMS).

It is well known that LMS is a powerful oxidative solution and it could be utilized in a wide range of biotransformations such as delignification and bleaching of paper pulps, detoxification of

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INTRODUCTION

pollutants or decolorization of industrial dyes (Kunamneni et al. 2008; Cañas and Camarero 2010). Upon the oxidation by laccase, the mediator oxidizes the substrate returning to its initial form, thereby closing the redox cycle, Figure 1.2. Fundamental characteristics required for an ideal mediator are that it must be a good laccase substrate, must be stable in both of its oxidation states, must not be an enzyme inhibitor and its redox conversion must be cyclic (Morozova et al.

2007). It is crucial that redox mediator is not eliminated from the reaction by secondary chemical transformations after one or several cycles. Also, it is important to emphasize that, as laccases, mediators with a high-redox potentialwill likely react more efficiently with high-redox potential substrates than low-redox potential mediators (Rochefort, Leech, and Bourbonnais 2004) Unfortunately, majority of high-redox potentialmediators are bad substrates for laccases because reaction is not thermodynamically favorable – due to their difference between redox potentials.

All these constraints heavily limit the number of compounds that could be used as a mediator.

Figure 1.2 Schematic representation of Laccase mediator system.

The first synthetic compound to be demonstrated as laccase mediator for the oxidation of nonphenolic lignin models was ABTS (Bourbonnais and Paice 1990). The first stage in ABTS oxidation generates the cationic radical (ABTS^·+) which is slowly oxidized to the dication (ABTS²⁺), eventually acting on the substrates following pure electron transfer mechanism (ET) (Morozova et al. 2007). Given their high redox potential and radical nature, the most effective laccase mediators for the oxidation of recalcitrant aromatic compounds are synthetic N–OH mediators such as HBT, violuric acid (VLA), N-hydroxyphthalimide (HPI), or N- hydroxyacetanilide (NHA) (Call 1994; Paice et al. 1997; Srebotnik and Hammel 2000; Xu et al.

2000). Upon oxidation by laccase, they form radical cations which undergo spontaneous deprotonation to highly active aminoxyl radicals. The latter can remove hydrogen atom from high redox potential substrates, in so-called hydrogen-atom transfer (HAT) route (Galli and Gentili 2004). However, their radical nature causes stability problem in the redox cycle which can result in rapid decomposition even inhibiting laccase activity (Li, Xu, and Eriksson 1999; Rochefort, Leech, and Bourbonnais 2004). In order to improve LMS for pulp delignification, Rochefort and

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INTRODUCTION

results as HBT, a much more reactive radical forming mediator. Apart from its high redox potential (E = +780 mV vs. NHE), K4[Mo(CN)8] does not generate radicals when it is oxidized by laccase while it does follow the ET route. Although ET reactions with lignin are slower than reactions with radicals, transition metal coordination complexes proved to be very efficient mediators due to their strong stability. One important aspect of this Thesis will focus on the use of chemically diverse mediators to screen laccase mutant libraries constructed by laboratory evolution in order to enhance ET1 and how such modifications can be transferred to the use of LMS for the efficient oxidation of recalcitrant compounds, Figure 1.3.

Figure 1.3 Structures of redox mediators employed in this Thesis.

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INTRODUCTION

1.2 Industrial and biotechnological applications

The broad substrate specificity, use of air and generation of water as the only by-product make laccases ideal green biocatalysts (Riva 2006; Rodgers et al. 2010). In the last two decades great interest have emerged to use laccase in replacing standard chemical processes in the textile, pulp and paper and pharmaceutical industries. Furthermore, laccase have possible application in bioremediation processes, production of biofuels, in cosmetic, paint, food and furniture industries (Mate and Alcalde 2017). Also, due to their ability of direct electron transfer from cathodic compartment, laccases have been tightly related to the engineering of biosensors and biofuel cells (Falk et al. 2014; Rodríguez-Delgado et al. 2015). Figure 1.4 highlights the biotechnological potential of laccases sorted by the area of application.

Figure 1.4 Breakdown in the biotechnological applications of laccases. Data extracted from Scopus database search for articles that included the following keywords: (i) ‘laccase’ and ‘bioremediation’; (ii)

‘laccase’ and ‘biofuel cell’ or ‘biosensor’; (iii) ‘laccase’ and ‘textiles’ or ‘textiles industry’; (iv) ‘laccase’

and ‘pulp and paper’ or ‘pulp and paper industry’; (v) ‘laccase’ and ‘food’ or ‘food industry’; (vi) ‘laccase’

and ‘organic synthesis’; (vii) ‘laccase’ and ‘biofuel production’; (viii) ‘laccase’ and ‘fiberboards’; (ix)

‘laccase’ and ‘cosmetics’; (x) and ‘laccase’ and ‘paints’. Figure adapted and updated from (Mate and Alcalde 2017).

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INTRODUCTION

Some examples of laccase utility in various industrial fields are given below:

 Bioremediation: removal of PAHs such as anthracene or benzopyrene (Majcherczyk, Johannes, and Hüttermann 1998; Cañas et al. 2007; Zumárraga, Plou, et al. 2007; Zeng et al. 2016), and recalcitrant dyes like Reactive Black 5 or crystal violet aided by LMS (Camarero et al. 2005; Wang et al. 2016).

 Organic synthesis: synthesis of complex medical products, such as antibiotics (e.g.

penicillin X dimer and cephalosporins), anti-cancer drugs (e.g. vinblastine, mitomycin and actinomycin) and immunosuppressors (e.g. cyclosporin A) (Kunamneni et al. 2008);

enzymatic derivatization of amino acids, such as L-lysine, L-tryptophane and L- phenylalanine (Mogharabi and Faramarzi 2014); oxidation of steroid hormones 17b- estradiol and stilbenic phytoalexin trans-resveratrol (Nicotra, Cramarossa, et al. 2004;

Nicotra, Intra, et al. 2004).

 Pulp and paper industry: bleaching of flax pulp with laccases using redox natural and synthetic mediators (Camarero et al. 2002; Fillat, Colom, and Vidal 2010); grafting of phenols to flax fibres for paper production (Aracri et al. 2010; Fillat et al. 2012).

 Food industry: selective removal of polyphenols by laccases, hence avoiding undesired modifications of the wine’s organoleptic properties (Osma, Toca-Herrera, and Rodríguez-Couto 2010); immobilized laccase from T. versicolor has been recently used in clarification of fruit juice (de Souza Bezerra et al. 2015).

 Textile industry: bleaching of denim and cotton fabrics (Tzanov et al. 2003; Yavuz, Kaya, and Aytekin 2014); synthesis of C-N heteropolymeric dye by M. thermophila laccase (Vicente et al. 2016).

 Paint industry: employing LMS in chemical drying of alkyd resins widely used in different coatings and paints (Greimel et al. 2013).

 Cosmetics industry: in replacing H2O2 as oxidizing agent in the formulation of hair dyes.

Laccases from the actinomycete Thermobifida fusca and from the basidiomycete Flammulina velutipes have been tested in the oxidation of dye intermediates extensively used in hair coloring (Chen et al. 2013; Saito et al. 2012).

 Biofuel production: improving bioethanol production yields from lignocellulosic material by removing phenolic compounds which are inhibiting yeast growth (Larsson, Cassland, and Jönsson 2001; Fang et al. 2015)

 Biosensors: detection of polyphenols in wine, fruit juices and teas and quantification of fungal contamination in grape musts by employing laccase-based sensors (Zouari, Romette, and Thomas 1988; Ghindilis, Gavrilova, and Yaropolov 1992; Cliffe et al. 1994;

Di Fusco et al. 2010); laccase-based biosensors have also been used to detect insulin, morphine and codeine (Bauer et al. 1999; Milligan and Ghindilis 2002)

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INTRODUCTION

 Biofuel cells: laccases have been used as cathodic enzyme in the development of self- powered wireless carbohydrate/oxygen sensitive biodevice, opening new prospect in the application of implantable biofuel cells for medical devices and environmental monitoring (Falk et al. 2014).

1.3 Laccase engineering

1.3.1 General strategies

The demonstrated potential of laccases in a range of biotechnological applications has propelled the progress of laccase engineering efforts. Indeed, laccases became the focus of many attempts to adapt them to harsh industrial standards. Both fungal and bacterial laccases have been subjected to extensive engineering efforts via rational design and directed evolution (Mate and Alcalde 2015). Apart from engineering laccases to study their redox potential which was described in previous section, much effort has been done to improve or create other enzymatic features, such as: functional heterologous expression (Bulter et al. 2003; Koschorreck, Schmid, and Urlacher 2009; Mate et al. 2010; Camarero et al. 2011), activity (Madzak et al. 2005;

Zumárraga et al. 2008; Kataoka et al. 2013; Toscano et al. 2013; Pardo et al. 2016; Santiago et al.

2016), resistance to high temperatures (Mollania et al. 2011; Scheiblbrandner et al. 2017) or extreme pH (Torres-Salas et al. 2013), performance in non-natural environments like organic solvents (Zumárraga, Bulter, et al. 2007; Rasekh et al. 2014), human blood (Mate, Gonzalez- Perez, Falk, et al. 2013) or ionic liquids (Liu et al. 2013). The majority of these examples have been performed by means of directed evolution, an extremely potent strategy to design customized enzymes for different purposes by mimicking in the lab the processes of natural evolution. Genetic diversity is created by means of random mutagenesis and/or recombination of parental genes. After the protein is expressed in a suitable host organism, mutant libraries are screened towards specific traits applying a selective pressure which is under the strict control of the researcher, Figure 1.5. This procedure is repeated as many times as necessary until the desired property is achieved. In recent years, the combination of directed evolution and computational approaches is becoming possibly the most promising strategy in protein engineering. Certainly, in silico predictions by computational tools can help to reduce the exploration of the vast protein sequence space, to open new avenues in laccase design while reducing substantially the experimental efforts (Mate and Alcalde 2015; Molina-Espeja et al. 2016). This Thesis is bringing together different computational strategies and directed evolution methods aimed at optimizing

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INTRODUCTION

Figure 1.5 Schematic representation of a standard directed evolution cycle comprising three main steps: generation of genetic diversity, expression of the mutant library and screening of the best mutants

`winners´.

1.3.2 Functional expression of fungal laccases

To make directed enzyme evolution, the functional expression of the target enzyme in a suitable heterologous host is an indispensable requirement. In the case of fungal laccases, one of the main obstacles is their poor functional expression in a foreign host and/or limited secretion.

Although in theory any organism might serve as a host for engineering effort, in reality only few have been used (Pourmir and Johannes 2012). Filamentous fungi have exhibited the highest heterologous expression of fungal laccases reported to date, up to 900 mg/L (Baker and White 2001; Kiiskinen et al. 2004; Mekmouche et al. 2014); however, they are extremely impractical host for directed evolution (Viviane et al. 2011; Pourmir and Johannes 2012). On the other hand, Saccharomyces cerevisiae has been traditionally the chosen expression host to improve fungal laccases by directed evolution owing to its high transformation efficiency -yielding thousands of individual colonies, in the range of 107-108 CFU per g of DNA-, the availability of episomal vectors and its simple fermentation requirements (Gonzalez-Perez, Garcia-Ruiz, and Alcalde 2012). More significantly, by exhibiting a high frequency of homologous DNA recombination, this yeast enables us to carry out a wide array of genetic manipulations, facilitating the generation of molecular diversity required for library creation methods. Also, it enables direct protein

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INTRODUCTION

secretion into the extracellular medium simplifying the detection of laccase activity without complex lysis steps. Different protein engineering strategies have been employed in directed evolution campaigns to achieve laccase functional expression in S. cerevisiae: i) directed evolution of mature laccase and its native signal peptide simultaneously (Bulter et al. 2003); ii) the replacement of the native signal peptide with different prepro-leaders and its joint evolution with the mature laccase (Mate et al. 2010; Camarero et al. 2011); and iii) focus mutagenesis only on the signal peptide or just on mature protein. The main advantage of the former approach is that only the protein secretion is under selection, while in the latter mutations introduced in mature protein could elevate secretion but also modify activity, making difficult the breakdown between both factors.

1.3.3 Laccase chimeragenesis

A particular promising strategy in laccase engineering is the design of chimeras with combined properties by DNA recombination methods. In general terms, mutations introduced by homologous recombination are often less deleterious than random mutations because they are compatible with the backbone structure (Meyer, Hochrein, and Arnold 2006). However, considering the complex structural organization of laccase with two copper centers and an intramolecular electron transfer between them, the generation of chimeras with high sequence diversity from different orthologs is difficult to be achieved without jeopardizing protein functionality. For that reason, while laboratory evolution campaigns from one single laccase template proved to be successful, limited results have been accomplished in attempts of creating laccase chimeras from different organisms. For example, when it was tried the engineering of hybrid enzymes from two high-redox potential laccases (evolved laccase versions from basidiomycetous PM1 and Pycnoporus cinnabarinus) towards functional expression in yeast (Mate et al. 2010; Camarero et al. 2011) by in vitro and in vivo DNA shuffling, only a set of functional chimeras with low sequence diversity -i.e. mostly enriched by the parent with the highest expression and showing few crossover events in mature protein-, was obtained (Pardo et al. 2012). Similarly, chimeric laccases designed by in vivo shuffling in S. cerevisiae of several isoforms (Lac1, Lac2, Lac3 and Lac5 from Trametes sp. strain C30) led to a library primed to the Lac3 parent due to its higher expression levels in the heterologous host (Cusano et al. 2009). Also, a preliminary rational design of chimeras was attempted with two isoforms (Lcc1 and Lcc4) from basidiomycete Lentinula edodes expressed in tobacco By-2 cells where the N-terminus of lcc1 was connected to the C-terminus of the Lcc4 (i.e. producing only one crossover point, (Nakagawa et al. 2010)). Certainly, obtaining significant sequence and functional diversity from laccase

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INTRODUCTION

1.3.3.1 SCHEMA-RASPP computational algorithm

SCHEMA structure-guided protein recombination is a computational method proved to be extremely useful when creating chimeric proteins with great sequence diversity improving most notably thermostability, but as well, substrate specificity, pH dependence and secretion (Li et al. 2007; Heinzelman et al. 2009; Heinzelman et al. 2010). This approach has been employed most extensively with cytochrome P450 monooxygenases, but as well as with β-lactamases, cellulases, arginases and channelrhodopsins; however, it has never been used to create chimeric laccases (Otey et al. 2004; Meyer, Hochrein, and Arnold 2006; Otey et al. 2006; Li et al. 2007;

Heinzelman et al. 2009; Heinzelman et al. 2010; Romero et al. 2012; Smith et al. 2012; Bedbrook et al. 2017). SCHEMA identifies protein blocks that can be recombined by selecting suitable crossover locations in contiguous sequence that minimize the schema energy <E> – the library average number of interactions that are broken upon recombination of different parents, Figure 1.6, (Voigt et al. 2002). Two residues are considered interacting if any of their atoms (hydrogen excluded) are within a cutoff distance of 4.5 Å, Figure 1.7. Basically, SCHEMA predicts protein blocks that must be inherited from the same parent for hybrid protein to be functional. To complement the SCHEMA method, Arnold´s group also developed RASPP (Recombination as Shortest Path Problem) algorithm which generate libraries by minimizing <E> for a range of <m>

– average number of mutations in library relative to the closest parent, enabling to create functional chimeras with high sequence diversity (Endelman et al. 2004). RASPP algorithm generates optimized libraries with varying degrees of diversity by adjusting limitations on block length and establishing minimum number of mutations in each block. Then, comparing libraries that RASPP generate, we can select a library with good balance between the schema energy and the average number of mutations.

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INTRODUCTION A B

Figure 1.6 Representation of SCHEMA approach. (A) DNA level. (B) Protein level. Parental genes and proteins are depicted in different colors. After fragmentation at exact crossover locations determined by computational algorithm, blocks are randomly recombined yielding functional chimeric proteins with combined or improved properties.

Figure 1.7 Representation of SCHEMA disruption. Each residue in the structure is represented as a sphere. Black lines represent peptide bond and green dashed lines represent interaction between amino acid side chains. Two chimeric proteins are shown. In the case of offspring 1, the seven last residues are inherited from parent 2 (in red) and the rest of the structure from parent 1 (in blue), two interactions are broken (<E>

= 2) leading to the unfolded hybrid protein. On the other hand, when the last four residues are inherited from parent 2, there is no disruption and chimeric protein is folded and functional.

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2 OBJECTIVES

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2 OBJECTIVES

The first objective of this Thesis was to enhance the redox potential at T1Cu site (ET1) of a HRPL by means of computer-guided mutagenesis and directed evolution. The following tasks were carried out:

1) Design and optimization of ad-hoc screening assays based on the oxidation of high-redox potential mediators.

2) Generation of mutagenic libraries using protein engineering techniques aided by computational simulations.

3) Biochemical, spectroelectrochemical and computational characterization of the final mutant variant.

The second objective of this Thesis was to generate novel fungal chimeric laccases using SCHEMA-RASPP structure-guided recombination in vivo. Laccases from PM1 (OB-1 variant), Pycnoporus cinnabarinus (3PO variant) and Trametes C30 sp. (Lac3 variant) were employed for this purpose. The following tasks were carried out:

1) Analysis of the secretion levels of parental laccases using different prepro-leaders in order to select the signal peptide conferring the highest secretion yields.

2) Design a chimeric library enriched in sequence and functional diversity by SCHEMA- RASPP computational algorithm in combination with in vivo shuffling.

3) Screening of the chimeric library at high temperature and biochemical study of the resulting family of functional chimeras.

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3 MATERIALS AND METHODS 3 MATERIALS AND

METHODS

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MATERIALS AND METHODS

3.1 Reagents and materials

Table 3.1.1 Reagents.

Reagents Company

1-hydroxybenzotriazole (HBT) Sigma

2, 6-dimethoxyphenol (DMP) Sigma

2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

(ABTS) Sigma

Agarose BioRad

Biotin Sigma

Bis-Tris Fluka

Calcium suplhate dihydrate sigma

Copper (II) sulfate pentahydrate Sigma

Ethanol Panreac

Dimethyl sulfoxide (DMSO) Sigma

Glycerol Panreac

Guaiacol Sigma

Magnesium chloride Sigma

Magnesium sulfate heptahydrate Sigma

Manganese (II) chloride Sigma

Methanol Sigma

Phosphoric acid Sharlau

Poly R-478 Sigma

Potassium hydroxide Panreac

Potassium octacyanomolybdate Sigma

Potassium phosphate monobasic Sigma

Potassium sulfate Sigma

PTM1 trace salts Invitrogen

Reactive black 5 Sigma

Sinapic acid Sigma

Violuric acid Sigma

β-Mercaptoethanol Sigma

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Table 3.1.2 Medium components.

Medium components Company

Amino acids supplements without uracil Difco (BD)

Ampicilin Sigma

Antifoam 204 Sigma

Bacto Agar BD

Bacto peptone BD

Chloramphenicol Sigma

D-(+)-Galactose Sigma

D-(+)-Glucose Sigma

D-(+)-Rafinose pentahydrate Sigma

Uracil Sigma

Yeast extract BD

Yeast nitrogen base without amino acids (YNB) Difco (BD)

Zeocin Invitrogen

Table 3.1.3 Molecular biology kits.

Molecular biology kits Company

Deoxyribonucleotide triphosphate (dNTP) Sigma

DNA ligation kit New England

Biolabs

Gel Loading Solution Sigma

Gel Red DNA stain BioRad

GeneRuler 1Kb DNA Ladder Thermo Scientific

GeneRuler 100pb Plus DNA Ladder Thermo Scientific

NucleoSpin plasmid® Kit Macherey Nagel

Protein assay dye reagent Kit II (Bradford) BioRad

Yeast Transformation Kit Sigma

Zymoclean Gel DNA recovery Kit Zymoresearch

Zymoprep Yeast Plasmid Miniprep Kit Zymoresearch

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Table 3.1.4 Strains and plasmids.

Strains and plasmids Company

Escherichia coli XL2-Blue competent cells Stratagene

Expression shuttle vector pJRoC30 Caltech

Pichia pastoris expression vector (pPICZ B) Invitrogen

Pichia pastoris strain X-33 Invitrogen

S. cerevisiae strain BJ5465 LGC Promochem

Table 3.1.5 Commercial enzymes.

Commercial enzymes Company

Antarctic phosphatase New England Biolabs

Bovine serum albumin (BSA) Sigma

Mutazyme II Agilent

Pfu-ultra High Fidelity DNA Polymerase Agilent

Taq DNA Polymerase Sigma

BamHI restriction enzyme New England Biolabs

BstI restriction enzyme New England Biolabs

SacI restriction enzyme New England Biolabs

XbaI restriction enzyme New England Biolabs

XhoI restriction enzyme New England Biolabs

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3.2 Culture media

Composition of all mediums described in this chapter is referred to a final volume of one liter of distilled water. All mediums and solutions, if not specified otherwise, were autoclaved 30 min at 121°C.

3.2.1 Culture media for bacteria (E. coli) growth

Table 3.2.1 Luria-Bertani medium with ampicillin (LB/Amp).

Bacto Peptone 10 g

Yeast Extract 5 g

NaCl 10 g

Ampiciline (100 mg/mL) 1 mL

LB/Amp medium for the selective growth of E. coli cells transformed with pJRoC30 vector which contains ampicillin resistance gene (Sambrook, Fritsch, and Maniatis 1989). First, extracts and salt were dissolved in distilled water and pH was adjusted to 7.0. After the sterilization by autoclaving it was necessary to wait until temperature decrease to 50°C and thereafter, filtered ampicillin was added. In order to prepare solid medium, 20 g of agar were added before sterilization.

Table 3.2.2 Super optimal broth medium (SOB).

Bacto Peptone 2 g

Yeast Extract 0,5 g

NaCl 0,05 g

KCl 1 mL

Extracts and salts were dissolved in distilled water, pH was adjusted to 7.0 and sterilized by autoclave.

Table 3.2.3 Super optimal broth medium with catabolite repression (SOC).

SOB medium 5 mL

MgCl2 (2 M) 25 µL

Glucose (20% p/v) 100 µL

SOC medium is used for E. coli transformation (Sambrook, Fritsch, and Maniatis 1989). MgCl2

and glucose were previously filter-sterilized and thereafter mixed with SOB medium. For each transformation it was necessary to prepare a new SOC solution.

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3.2.2 Culture medium for yeast (S. cerevisiae and P. pastoris) growth

Table 3.2.4 YP medium (1.55 x).

Bacto Peptone 30,77 g

Yeast Extract 15,38 g

YP medium is used in laccase expression medium. The components were dissolved in water and the medium was sterilized by autoclave.

Table 3.2.5 YPD medium.

Bacto Peptone 20 g

Yeast Extract 20 g

Glucose (20% p/v) 100 mL

Chloramphenicol (25 mg/mL) 1 mL

YPD medium for yeast growth. After the components were dissolved in water, the medium was sterilized by autoclave. After sterilization it was necessary to wait until temperature decrease to 50°C and thereafter, filtered glucose and chloramphenicol were added. In order to prepare solid medium, 20 g of agar were added before sterilization.

Table 3.2.6 Minimal liquid medium.

YNB medium (67 g/L) 100 mL

Amino acids Supplements (10 x) 100 mL

Rafinose (20% p/v) 100 mL

Selective medium without uracil for the growth of S. cerevisiae cells transformed with pJRoC30 (+ gene), which contains gene ura3 that complements for uracil auxotrophy. Water is previously autoclaved and after the temperature decrease to 50°C the remaining components -previously sterilized by filtration were added.

Table 3.2.7 Minimal solid medium (SC drop-out plates).

Bacto agar 20 g

YNB médium (67 g/L) 100 mL

Amino acids Supplements (10 x) 100 mL

Glucose (20% p/v) 100 mL

Selective medium without uracil for the growth of S. cerevisiae cells transformed with pJRoC30 (+ gene), which contains gene ura3 that complements for uracil auxotrophy. It is prepared as described above for the minimal liquid medium, but including agar in the autoclave sterilization.

Directed and computational evolution of fungal laccases: redox potential enhancement and development of a family of thermostable chimeras

IVAN MATELJAK

TESIS DOCTORAL

Madrid, 2018

ACKNOWLEDGEMENTS

SUMMARY

RESUMEN

CONTENTS

ACRONYMS

1 INTRODUCTIO N

1 INTRODUCTION

1.1 Laccases

1.2 Industrial and biotechnological applications

1.3 Laccase engineering

2 OBJECTIVES

2 OBJECTIVES

3 MATERIALS AND METHODS 3 MATERIALS AND

METHODS

3.1 Reagents and materials

3.2 Culture media