Moreno-Giménez Elena, Gandía Mónica, Sáez Zara, Manzanares Paloma, Yenush Lynne, Orzáez Diego, Marcos Jose F, Garrigues Sandra
Food Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain.
Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC)-Universitat Politècnica de València (UPV), Valencia, Spain.
Front Bioeng Biotechnol. 2023 Aug 7;11:1222812. doi: 10.3389/fbioe.2023.1222812. eCollection 2023.
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 -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 . Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker We also used a normalized luciferase reporter system to validate several promoters, such as P, P, Pα, and P constitutive promoters, and P, P, and P 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.
真菌合成生物学是一个迅速发展的领域，旨在通过生成标准的、现成可用的遗传元件以及通用的语法和规则，来优化真菌的生物技术利用，以供真菌研究群体共同使用。最近，越来越多的合成生物学工具包已被开发并应用于丝状真菌，这凸显了这些生物体在生物技术领域的重要性。真菌编织（FungalBraid，FB）模块化克隆平台能够使DNA片段与为植物设计的金辫子（GoldenBraid，GB）平台以及其他与标准金门克隆和语法兼容的系统实现互换，并使用二元pCAMBIA衍生载体来实现多种真菌物种的农杆菌介导转化。在本研究中，我们通过添加27个经过功能验证的新DNA片段，扩展了原始的FB文库。其中包括针对抗生素博来霉素和特比萘芬的抗性选择标记，以及尿苷营养缺陷型标记。我们还使用了标准化的荧光素酶报告系统来验证多个启动子，如组成型启动子P、P、Pα和P，以及诱导型启动子P、P和P。此外，最近开发的用于植物正交CRISPR激活（CRISPRa）的dCas9调控GB_SynP合成启动子文库已通过FB系统在真菌中进行了适配。总体而言，FB文库的扩展对科学界具有重大意义，因为它增加了可能的模块化和可互换DNA组装的数量，成倍增加了研究、开发和利用丝状真菌的可能性。
