FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

Elena Moreno-Giménez; Mónica Gandía; Zara Sáez; Paloma Manzanares; Lynne Yenush; Diego Orzáez; Jose F Marcos; Sandra Garrigues

doi:10.3389/fbioe.2023.1222812

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

Front Bioeng Biotechnol. 2023 Aug 7:11:1222812. doi: 10.3389/fbioe.2023.1222812. eCollection 2023.

Authors

Elena Moreno-Giménez^{1

2}, Mónica Gandía³, Zara Sáez¹, Paloma Manzanares¹, Lynne Yenush², Diego Orzáez², Jose F Marcos¹, Sandra Garrigues¹

Affiliations

¹ Food Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain.
² Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC)-Universitat Politècnica de València (UPV), Valencia, Spain.
³ Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department. Faculty of Pharmacy. Universitat de València. Vicente Andrés Estellés s/n, Valencia, Spain.

Abstract

Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, ready-to-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciens-mediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA, P7760, Pef1α, and PafpB constitutive promoters, and PglaA, PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.

Keywords: CRISPR activation; GoldenBraid; filamentous fungi; fungal synthetic biology; luciferase-based reporter system; promoters; selection markers.

Grants and funding

This work was supported by PROMETEO/2018/066 from “Conselleria d’Educació” (Generalitat Valenciana, Comunitat Valenciana, Spain), grant PID 2021-125858OB-100, and the Severo Ochoa Excellence Program CEX 2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe.” EM-G was the recipient of a predoctoral grant FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future.” SG holds a Juan de la Cierva Incorporación grant (IJC 2020-042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR.