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). 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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. 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See above.https://gbcloning.upv.es/https://benchling.co

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology

[EN] Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. BIO2010-15384), by a Research Personnel in Training fellowship to A.S.-P., and by a Junta de Ampliacion de Estudios fellowship to M.V.-V.Sarrion-Perdigones, A.; Vázquez Vilar, M.; Palací Bataller, J.; Castelijns, B.; Forment Millet, JJ.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiology. 162(3):1618-1631. https://doi.org/10.1104/pp.113.217661S16181631162

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (alpha 1 and alpha 2) with one of the two LC types (k and lambda) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HC alpha 1 and LC lambda provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 mu g IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.This work has been funded by Grant BIO2010-15384 from Plan Nacional I + D of the Spanish Ministry of Science. Juarez P. is a recipient of a FPU fellowship, and Sarrion-Perdigones A. and Huet-Trujillo E. are recipients of a FPI fellowship. We want to thank Monedero for kindly providing scFv and VP8* clones.Juárez Ortega, P.; Huet Trujillo, E.; Sarrion-Perdigones, A.; Falconi, E.; Granell Richart, A.; Orzáez Calatayud, DV. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences. 14(3):6205-6222. https://doi.org/10.3390/ijms14036205S62056222143Reichert, J. M., & Valge-Archer, V. E. (2007). 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GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

This is a pre-copyedited, author-produced version of an article accepted for publication in Nucleic Acids Research following peer review. The version of record Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernandez Del Carmen, MA.; Sarrion-Perdigones, A.; Ochoa-Fernández, R.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Research. 45(4):2196-2209. doi:10.1093/nar/gkw1326 is available online at: http://doi.org/10.1093/nar/gkw1326.[EN] Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.].Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernández Del Carmen, MA.; Sarrion-Perdigones, A.; Ochoa-Fernández, R.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. 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Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp

[EN] Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Método de producción de repertorios complejos de moléculas recombinantes

La presente invención describe un método de producción de repertorios complejos de moléculas recombinantes de forma consistente y reproducible, mediante la generación de forma transitoria de plantas multi-transgénicas que dan lugar a mosaicos somáticos inducidos por replicones virales que se excluyen entre sí. La presente invención también hace referencia a la planta multi-transgénica o un fragmento de la misma así obtenida, así como a los extractos o fracciones purificadas de las mismas que representen repertorios complejos de moléculas recombinantes, preferentemente proteínas recombinantes seleccionadas entre: enzimas, inmunoglobulinas, receptores de membrana, receptores intracelulares, lectinas, anticuerpos policlonales, antivenenos basados en antisueros, sueros inmunológicos para inmunidad pasiva, e inmunoglobulinas intravenosasPeer reviewedConsejo Superior de Investigaciones Científicas. Universidad Politécnica de Valencia, Universidad de la Palmas de Gran CanariaB1 Patente sin examen previ

Método de producción de repertorios complejos de moléculas recombinantes

La presente invención describe un método de producción de repertorios complejos de moléculas recombinantes de forma consistente y reproducible, mediante la generación de forma transitoria de plantas multi-transgénicas que dan lugar a mosaicos somáticos inducidos por replicones virales que se excluyen entre sí. La presente invención también hace referencia a la planta multi-transgénica o un fragmento de la misma así obtenida, así como a los extractos o fracciones purificadas de las mismas que representen repertorios complejos de moléculas recombinantes, preferentemente proteínas recombinantes seleccionadas entre: enzimas, inmunoglobulinas, receptores de membrana, receptores intracelulares, lectinas, anticuerpos policlonales, antivenenos basados en antisueros, sueros inmunológicos para inmunidad pasiva, e inmunoglobulinas intravenosasPeer reviewedConsejo Superior de Investigaciones Científicas. Universidad Politécnica de Valencia, Universidad de la Palmas de Gran CanariaA1 Solicitud de patente con informe sobre el estado de la técnic

Método de producción de repertorios complejos de moléculas recombinantes

[EN] The invention relates to a method for the production of complex repertoires of recombinant molecules in a consistent and reproducible manner, comprising the transient generation of multi-transgenic plants that generate somatic mosaics induced by viral replicons that are mutually exclusive. The invention also relates to the multi-transgenic plant or a fragment of same obtained in this way, as well as to extracts or purified fractions thereof, which represent complex repertoires of recombinant molecules, preferably recombinant proteins selected from among: enzymes, immunoglobulins, membrane receptors, intracellular receptors, lectins, polyclonal antibodies, antidotes based on anti-serums, immunological serums for passive immunity, and intravenous immunoglobulins[ES] La presente invención describe un método de producción de repertorios complejos de moléculas recombinantes de forma consistente y reproducible, mediante la generación de forma transitoria de plantas multi-transgénicas que dan lugar a mosaicos somáticos inducidos por replicones virales que se excluyen entre sí. La presente invención también hace referencia a la planta multi-transgénica o un fragmento de la misma así obtenida, así como a los extractos o fracciones purificadas de las mismas que representen repertorios complejos de moléculas recombinantes, preferentemente proteínas recombinantes seleccionadas entre: enzimas, inmunoglobulinas, receptores de membrana, receptores intracelulares, lectinas, anticuerpos policlonales, antivenenos basados en antisueros, sueros inmunológicos para inmunidad pasiva, e inmunoglobulinas intravenosasPeer reviewedConsejo Superior de Investigaciones Científicas. Universidad Politécnica de Valencia, Universidad de la Palmas de Gran CanariaA1 Solicitud de patente con informe sobre el estado de la técnic

Sistema para ensamblado de piezas genéticas

La presente invención describe un sistema de ensamblado in vitro para piezas genéticas. El ensamblado de fragmentos de DNA constituye la base de la ingeniería genética y la biología sintética. Para el diseño de nuevos circuitos genéticos, ambas disciplinas tienden hacia la generación de colecciones de piezas genéticas intercambiables y reciclables (es decir, susceptibles de ser utilizadas en laboratorios distintos y para generar combinaciones genéticas distintas), que puedan ser unidas entre sí mediante el uso de un método estándar de ensamblaje. Es particularmente necesario el desarrollo de métodos que permitan gran eficiencia y versatilidad en el ensamblaje de piezas en los rangos que van entre 5 y 50 piezas individuales, ya que la naturaleza modular de las interacciones genéticas hace que buena parte de la ingeniería se desarrolle en torno a diseños genéticos que abarcan estos rangos de tamaño.Peer reviewedConsejo Superior de Investigaciones Científicas, Universidad Politécnica de ValenciaA1 Solicitud de patente con informe sobre el estado de la técnic