Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity

Manuscript of the article published in print 13 April 2016. The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.jafc.6b00222 .Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3′-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3′-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.This work was funded by grant BIO2013-48779-C4-3-R, from Spain's 'Secretaría de Estado de Investigación, Desarrollo e Innovación'. D T-P was supported by a FPU fellowship from 'Ministerio de Economía y Competitividad'.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

Separation of an aqueous mixture of 6-kestose/sucrose with zeolites: A molecular dynamics simulation

[EN] Extra-large pore zeolites are a small subset (21) among the whole list of 253 zeolites available. The discovery of new low-glycemic sugars is very attractive as new healthy additives in the food field. This is the case of the 6-kestose. In the present case, it appears in a mixture in aqueous solution together with sucrose, the separation of the mixture being necessary. For this, we have focused on using certain zeolites with adequate pore sizes that allow the separation of this mixture, considering that since the molecular size of 6-kestose is greater than sucrose, it is necessary to promote the sorption of the latter, so that the first can be purified. After a computational screening of micropores of the 253 IZA zeolites, 11 zeolites were selected. Of these, 3 extra-large pore zeolites (AET, DON, ETR) have been proposed, which were analyzed in-depth through a molecular dynamics study considering the external surface. The results show that DON presents the most promising theoretical results for a selective sucrose/6-kestose separation.We thank MICINN of Spain for funding through projects RTI2018101784-B-I00, RTI2018-101033-B-I00, SEV-2016-0683 as well as ASICUPV and CESGA for computational facilities. IBL and PGI gratefully acknowledge CSIC for a JAE-Intro fellowship. AM thanks Generalitat Valenciana for the predoctoral fellowship GRISOLIAP/2019/084.Bolaño Losada, I.; Grobas-Illobre, P.; Misturini, A.; Polaina, J.; Seminóvski, Y.; Sastre Navarro, GI. (2021). Separation of an aqueous mixture of 6-kestose/sucrose with zeolites: A molecular dynamics simulation. Microporous and Mesoporous Materials. 319:1-10. https://doi.org/10.1016/j.micromeso.2021.111031S11031

Fixation of bioactive compounds to the cuticle of Artemia

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia, and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus. Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc., to the cuticle of Artemia as well as other arthropods. Statement of relevance: Our results overcome shortcomings of Artemia as a feedstock.En prensa2,04

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.This article is dedicated to the memory of our friend and colleague Dr. Alejandro Sarrion-Perdigones, an early developer of GoldenBraid. We acknowledge the excellent technical assistance provided by Marisol Gascón (IBMCP, Valencia, Spain) with the fluorescent images. This work was funded by Grant BIO2013- 42193 and Grant BIO2016-78601-R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018-101115-B-C21 from the Ministerio de Ciencia, Innovación y Universidades (Spain) (MICINN/FEDER Funds), and PROMETEO/ 2018/066 from Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018- 092893-ERA CoBioTech (109) (MCIU/FEDER).Vázquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.... (2020). Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Current Protocols in Molecular Biology. 130(1):1-31. https://doi.org/10.1002/cpmb.116S1311301Bernabé‐Orts, J. M., Casas‐Rodrigo, I., Minguet, E. G., Landolfi, V., Garcia‐Carpintero, V., Gianoglio, S., … Orzaez, D. (2019). Assessment of Cas12a‐mediated gene editing efficiency in plants. Plant Biotechnology Journal, 17(10), 1971-1984. doi:10.1111/pbi.13113Ballester, A.-R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., … Gabaldón, T. (2015). Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity. Molecular Plant-Microbe Interactions®, 28(3), 232-248. doi:10.1094/mpmi-09-14-0261-fiKhang, C. H., Park, S.-Y., Lee, Y.-H., & Kang, S. (2005). A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genetics and Biology, 42(6), 483-492. doi:10.1016/j.fgb.2005.03.004Chen, C., Liu, J., Duan, C., Pan, Y., & Liu, G. (2020). Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum. Fungal Genetics and Biology, 134, 103279. doi:10.1016/j.fgb.2019.103279Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression inSaccharomyces cerevisiae. Yeast, 26(10), 545-551. doi:10.1002/yea.1705Fräbel, S., Wagner, B., Krischke, M., Schmidts, V., Thiele, C. M., Staniek, A., & Warzecha, H. (2018). Engineering of new-to-nature halogenated indigo precursors in plants. Metabolic Engineering, 46, 20-27. doi:10.1016/j.ymben.2018.02.003Fresquet-Corrales, S., Roque, E., Sarrión-Perdigones, A., Rochina, M., López-Gresa, M. P., Díaz-Mula, H. M., … Cañas, L. A. (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLOS ONE, 12(9), e0184839. doi:10.1371/journal.pone.0184839Garrigues, S., Gandía, M., & Marcos, J. F. (2015). Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum. Applied Microbiology and Biotechnology, 100(5), 2243-2256. doi:10.1007/s00253-015-7110-3Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. doi:10.1038/nmeth.1318Gurgel, I. L. da S., Jorge, K. T. de O. S., Malacco, N. L. S. de O., Souza, J. A. M., Rocha, M. C., Fernandes, M. F., … Soriani, F. M. (2019). The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System. mSphere, 4(3). doi:10.1128/msphere.00350-19Hernanz-Koers, M., Gandía, M., Garrigues, S., Manzanares, P., Yenush, L., Orzaez, D., & Marcos, J. F. (2018). FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology. Fungal Genetics and Biology, 116, 51-61. doi:10.1016/j.fgb.2018.04.010Juarez, P., Huet-Trujillo, E., Sarrion-Perdigones, A., Falconi, E., Granell, A., & Orzaez, D. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences, 14(3), 6205-6222. doi:10.3390/ijms14036205Kramer, M. F., & Coen, D. M. (2001). Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb1501s56Marcet-Houben, M., Ballester, A.-R., de la Fuente, B., Harries, E., Marcos, J. F., González-Candelas, L., & Gabaldón, T. (2012). Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-646Michielse, C. B., J Hooykaas, P. J., J J van den Hondel, C. A. M., & J Ram, A. F. (2008). Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols, 3(10), 1671-1678. doi:10.1038/nprot.2008.154Müller, K. M., & Arndt, K. M. (2011). Standardization in Synthetic Biology. Synthetic Gene Networks, 23-43. doi:10.1007/978-1-61779-412-4_2Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., … Rogers, C. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. doi:10.1111/nph.13532Pérez-González, A., Kniewel, R., Veldhuizen, M., Verma, H. K., Navarro-Rodríguez, M., Rubio, L. M., & Caro, E. (2017). Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnology, 17(1). doi:10.1186/s12896-017-0393-yPérez-Nadales, E., & Di Pietro, A. (2011). The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum  . The Plant Cell, 23(3), 1171-1185. doi:10.1105/tpc.110.075093Salazar-Cerezo, S., Kun, R. S., de Vries, R. P., & Garrigues, S. (2020). CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer. Enzyme and Microbial Technology, 133, 109463. doi:10.1016/j.enzmictec.2019.109463Sarrion-Perdigones, A., Chang, L., Gonzalez, Y., Gallego-Flores, T., Young, D. W., & Venken, K. J. T. (2019). Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying. Nature Communications, 10(1). doi:10.1038/s41467-019-13651-ySarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., … Orzaez, D. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618-1631. doi:10.1104/pp.113.217661Selma, S., Bernabé‐Orts, J. M., Vazquez‐Vilar, M., Diego‐Martin, B., Ajenjo, M., Garcia‐Carpintero, V., … Orzaez, D. (2019). Strong gene activation in plants with genome‐wide specificity using a new orthogonal CRISPR /Cas9‐based programmable transcriptional activator. Plant Biotechnology Journal, 17(9), 1703-1705. doi:10.1111/pbi.13138Shendure, J. A., Porreca, G. J., Church, G. M., Gardner, A. F., Hendrickson, C. L., Kieleczawa, J., & Slatko, B. E. (2011). Overview of DNA Sequencing Strategies. Current Protocols in Molecular Biology, 96(1). doi:10.1002/0471142727.mb0701s96Szewczyk, E., Nayak, T., Oakley, C. E., Edgerton, H., Xiong, Y., Taheri-Talesh, N., … Oakley, B. R. (2006). Fusion PCR and gene targeting in Aspergillus nidulans. Nature Protocols, 1(6), 3111-3120. doi:10.1038/nprot.2006.405Vafaee, Y., Staniek, A., Mancheno-Solano, M., & Warzecha, H. (2014). A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors. PLoS ONE, 9(10), e110222. doi:10.1371/journal.pone.0110222Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2016). A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1). doi:10.1186/s13007-016-0101-2Villiers, B. R. M., Stein, V., & Hollfelder, F. (2009). USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction. Protein Engineering, Design and Selection, 23(1), 1-8. doi:10.1093/protein/gzp063Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Sarrion‐Perdigones et al. (2013). See above.Hernanz‐Koersetal. (2018). See above.https://gbcloning.upv.es/https://benchling.co

Structural Basis of the Inhibition of GH1 β-glucosidases by Multivalent Pyrrolidine Iminosugars

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other β-glucosidases with therapeutic relevance is discussed under the light of these observations.Ministerio de Economía y Competitividad CTQ2016-77270-R, BIO2016-76601-C3-3-R, AGL2016-75245-RJunta de Andalucía FQM-345Fondazione CR Firenze 2016/084

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

Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach

The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46=N, 2°59=W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.We gratefully acknowledge the financial support provided by the Spanish Ministry of Economy and Competitiveness (project CSD2007-00005), the European Community project MAGICPAH (FP7-KBBE-2009-245226), the European Regional Development Fund (ERDF), and the Government of Canada through Genome Canada, Ontario Genomics Institute, and Ontario Research Fund (2009-OGI-ABC-1405 and ORF-GL2-01-004). M.-E.G. thanks the CSIC for a JAE fellowship.Peer Reviewe