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Some of the mayor known unknowns of modern science deal with how lifeappeared on Earth and how from there it diversified into the different life forms present today. These questions delve into the deep past of our planet, where biology intermingles with geology and chemistry, to explore the origin of life. In addition, by learning how biological systems change over time, we can design novel biological machines based on this knowledge to fulfil unmet tasks.The main overarching theme addressed in this topic is evolution. Not because of much repeated is Theodosius Dobzhansky 1964’s statement less true: Nothing makes sense in biology except in the light of evolution. Understanding evolution will provide us with clues about the origin of life, and on the precise molecular mechanisms that operate in living beings and how they change in time. We will then be ready to attempt putting together these mechanisms in novel ways, paving the way for synthetic biology. Evolution is the single most overarching and one of the few, if not only, general principles in Biology.In this topic, we explore and discuss several aspects related to the study of evolution, as a fundamental and core discipline in Life Sciences, which must permeate different research areas in the coming years.

Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014., 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 frquency 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 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 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 Asp199 Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Granada (España) del 09 al 12 de septiembre de 2014., Hace diez años, se descubrió en el hongo basidiomiceto Agrocybe aegerita una enzima capaz de catalizar actividades que engloban aquellas de la cloroperoxidasa de Caldaryomices fumago (CPO) y las de las monooxigenasas del citocromo P450 [1,2]. Con el nombre de peroxigenasa inespecífi ca (unspecifi c peroxygenase, UPO, EC 1.11.2.1), esta versátil enzima [3] ha demostrado suplir las carencias de las anteriores, con una elevada efi ciencia y selectividad. La UPO es una enzima extracelular e independiente de cofactores como el NAD(P)H y otras enzimas auxiliares (sólo necesita H2O2 para trabajar), al contrario que las P450, lo que reduce el coste de su aplicación. Hemos sometido a esta peroxigenasa hemo-tiolada (cisteína como ligando axial) a evolución dirigida hacia expresión funcional en Saccharomyces cerevisiae [4]. Tras cinco ciclos, se obtuvo la variante PaDa-I, con unos niveles de secreción de ~8 mg/L. También ha sido sobre-expresada en Pichia pastoris, obteniendo ~230 mg/L (material sin publicar). En ambos casos, las propiedades y comportamiento son equivalentes a los de la enzima homóloga. Así, disponemos de una plataforma de fácil uso para continuar su mejora hacia aplicaciones de interés biotecnológico. En nuestro caso, estamos sometiendo a PaDa-I a evolución hacia mejora de su actividad hidroxilativa sobre naftaleno, en detrimento de su actividad oxidativa (material sin publicar). Esta última transforma el compuesto de interés, 1-naftol, en productos no deseados. La actividad oxidativa en la UPO parece estar muy relacionada con su estabilidad, pero los resultados obtenidos son esperanzadores, con una nueva variante que reduce cinco veces la actividad oxidativa (peroxidasa) manteniendo su capacidad de transferencia de oxígeno (actividad peroxigenasa).

Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014., Unspecific peroxygenase (UPO, EC.1.11.2.1) is a heme-thiolate peroxidase with exclusive mono(per)oxygenase activity and plenty of potential applications in organic synthesis. Fuelled by catalytic amounts of H2O2, UPO acts as a self-sufficient mono-oxygenase with a complex catalytic mechanism that joins the reactive intermediates of heme-peroxidases and P450s (“peroxide shunt” pathway). Thus, the versatile peroxide-dependent monooxygenase activity of UPO based on a two-electron oxygenation mechanism, permits an array of reactions to occur, among them: bromide oxidation, sulfoxidation, N-oxidation, aromatic peroxygenation, double bond epoxidation, hydroxylation of aliphatic compounds and ether cleavages [1, 2]. In a recent work, our laboratory performed the first protein engineering study carried out on UPO. After 5 generations of molecular evolution in Saccharomyces cerevisiae, we were able to produce a highly active, soluble and stable enzyme that is readily secreted in yeast [3]. With the view of taking advantage of this evolutionary platform, in this communication, the evolved UPO variant has been subjected to several rounds of neutral genetic drift to explore the substrate promiscuity and the evolvability of this versatile enzyme.

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 access

Trabajo presentado en el 13th European Workshop on Lignocellulosics and Pulp, celebrado en Sevilla (España) del 24 al 27 de junio de 2014, Versatile peroxidases (VPs) are promiscuous high-redox potential biocatalysts with broad substrate specificity. VPs are strongly inhibited by modest concentrations of hydrogen peroxide which hampers their application in different industrial settings. In the current work, a VP mutant evolved in our laboratory for functional expression was used as departure point to tailor oxidative stability. A high-throughput assay based on the analysis of the apparent half-life using different H2O2:enzyme molar ratios was designed and employed to explore mutant libraries. After only one round of directed evolution two variants were selected showing apparent half-life of 10- 23 min with a 3,000-fold H2O2:enzyme molar excess., This work was supported by European Commission Projects PEROXICATS (FP7-KBBE-2010-4-26537), INDOX (FP7-KBBE-2013-7-613549), COST-Action CM1303-Systems Biocatalysis; and the National Project EVOFACEL (BIO2010-19697).

Trabajo presentado en el XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Madrid (España) del 3 al 6 de septiembre de 2013., Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco, EC 4.1.1.39) is the most abundant enzyme in biosphere highly distributed in plants, algae and bacteria. This complex enzymatic system is responsible for the CO2 assimilation in biomass. Accordingly, rubisco catalyzes the fixation of CO2 into ribulose-1,5-bisphosphate (RuBP) generating two molecules of 3-phosphoglycerate (3PGA) through the Calvin cycle. Despite its important role for energy production in nature, rubisco is an extremely inefficient catalyst (with turnover rates from 2 to 13 s-1). In addition to be an extremely slow biocatalyst, its carboxylate activity is hampered by an unwanted oxygenase activity, which promotes the fixation of O2 into RuBP producing one molecule of 3PGA and one molecule of 2-phosphoglycolate (2PG) through the photorespiration. 2PG is considered a waste product since its recycling consumes energy and release CO2. In the last few years, rubisco has been subjected to intense study in order to modify its catalytic properties by directed evolution. The current study describes the cloning, functional expression and directed evolution of a rubisco gene in Escherichia coli to explore the catalytic promiscuity of this system., This research has been funded by Repsol S.A.

Trabajo presentado en el XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Madrid (España) del 3 al 6 de septiembre de 2013., [P09-18] Chloroperoxidase (CPO; EC 1.11.1.10) from the filamentous fungus Caldariomyces (Leptoxyphium) fumago is a heme-thiolate peroxidase with a huge catalytic versatility. Apart from the classical peroxidative mechanism which allows the enzyme to oxidize phenols and anilines, CPO is also capable of performing epoxidations, halogenations, hydroxylations or sulfoxidation coupling the reduction of hydrogen peroxide to water. Hence, CPO shows a remarkable potential for many biotechnological settings with especial emphasis in decontamination and organic syntheses. Even though numerous industrial processes have been patented with CPO as biocatalysts, the real exploitation of this enzymatic system has not been achieved yet. Among the main hurdles for the implementation of CPO are poor reaction yields, a strong inactivation by hydrogen peroxide and more significantly, the lack of suitable heterologous expression systems. Indeed, despite numerous efforts, all attempts carried out to achieve heterologous functional expression have failed providing in the best of the cases tiny secretion levels. To circumvent these obstacles the use of protein engineering by directed evolution is a valuable approach. The current communication describes our efforts to functionally express CPO in the budding yeast Saccharomyces cerevisiae. Seven different fusion genes were constructed combining different secretion leaders with the mature CPO whilst modifying the C-terminal region involved during folding at the post-translational stages. Subsequently, the best fusion gene was subjected to several rounds of directed evolution to improve secretion using an ad-hoc HTS assay. Designing expression platforms for CPO based on S. cerevisiae will allow us to tailor CPO with improved characteristics.

208 p.-75 fig.-42 tab.-2 anexos., VP (versatile peroxidases, EC 1.11.1.16) secreted by white‐rot fungi are involved in natural decay of lignin. VP combine the general catalytic features of other haem‐containing enzymes (in terms of substrate specificity and reaction mechanisms), such as the high‐redoxpotential ligninolytic peroxidases, LiP (lignin peroxidase) and MnP (manganese peroxidase), with those of peroxidases with a lower redox potential, such as HRP (horseradish peroxidase) and CiP (Coprinopsis cinerea peroxidase). Thus VP behaves as a generalist biocatalyst, readily oxidizing a variety of compounds. Unfortunately, VP has not been successfully functional expressed in any heterologous host, which limits its potential development. In this context, directed molecular evolution represents an elegant shortcut to tailor enzymes with improved features. By mimicking the Darwinist algorithm of natural selection through iterative steps of random mutagenesis and/or DNA recombination, the temporal scale of evolution can be collapsed from millions of years into months rather than weeks of bench work. We have engineered the VP from Pleurotus eryngii to be functionally expressed in Saccharomyces cerevisiae by directed evolution. Firstly, the optimization of culture conditions for functional expression and the engineering of a reliable high‐throughput screening assay were performed. Afterwards, a fusion gene containing the VP from P. eryngii and the α factor preproleader from S. cerevisiae was constructed and subjected to four rounds of directed evolution, achieving a level of secretion in S. cerevisiae of 21 mg/L. The evolved variant for expression (R4) harbored four mutations and increased its total VP activity 129‐fold over parent type along with a noticeable improvement of the catalytic efficiency at the haem channel oxidation site. Whilst the catalytic Trp was unaltered after evolution, the Mn2+ oxidation site was negatively affected by the mutations. The signal leader processing by the STE13 protease at the Golgi compartment changed as consequence of VP expression, retaining the additional N‐terminal sequence EAEA (Glu‐Ala‐Glu‐Ala) that enhanced secretion. The engineered N‐terminally truncated variants displayed similar biochemical properties to those of the non‐truncated counterpart in terms of kinetics, stability and spectroscopic features. Finally, we took advantage of the laboratory evolution platform set here to improve the thermostability of VP. Three additional cycles of evolution led to a more thermostable variant (2‐1B), harboring 3 stabilizing mutations. 2‐1B mutant showed a T50 8°C higher than parental type and the thermoactivity range was widened (from 30‐45°C for parent type to 30‐50°C for 2‐ 1B). Moreover, as a consequence of laboratory evolution, some unexpected side‐effects were detected. The enzyme’s stability at alkaline pHs was significantly increased retaining ~60 % of its residual activity at pH 9.0. In addition, the Km for H2O2 was enhanced up to 15‐fold while the catalytic efficiency was maintained. Mutations introduced in the course of evolution seemed to affect secretion, stability and activities by establishing new interactions with surrounding residues., Proyectos europeos (Ref. BIORENEW, NMP2‐CT‐2006‐026456), (Ref. 3D‐NANOBIODEVICE, NMP4‐SL‐2009‐229255) , (Ref PEROXICATS, FP7‐KBBE‐2010‐4) y (Ref. CASCAT, CM0701) y Proyecto del Plan Nacional (EVOFACEL, Ref: BIO2010‐19697)

[EN] Versatile peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due to a complex molecular architecture, with three catalytic sites and several oxidation pathways. To improve the VP resistance to H2O2, an evolved version of this enzyme was subjected to a range of directed evolution and hybrid strategies in Saccharomyces cerevisiae. After five generations of random, saturation, and domain mutagenesis, together with in vivo DNA recombination, several structural determinants behind the oxidative destabilization of the enzyme were unmasked. To establish a balance between activity and stability, selected beneficial mutations were introduced into novel mutational environments by the in vivo exchange of sequence blocks, promoting epistatic interactions. The best variant of this process accumulated 8 mutations that increased the half-life of the protein from 3 (parental type) to 35 min in the presence of 3000 equiv of H2O2 and with a 6 °C upward shift in thermostability. Multiple structural alignment with other H2O2-tolerant heme peroxidases help to understand the possible roles played by the new mutations in the overall oxidative stabilization of these enzymes., European Commission Projects (Peroxicats-FP7-KBBE-2010-4-26537; Indox-FP7-KBBE-2013-7-613549; COST-Action CM1303: Systems Biocatalysis) and the National Projects (Evofacel) [BIO2010-19697], (Dewry) [BIO2013-43407-R] and Hipop (BIO2011-26694). F.J.R.-D. is grateful for the award of a “Ramón y Cajal” contract of the Spanish MINECO.