Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

中文翻译：

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FungalBraid：基于GoldenBraid的模块化克隆平台，用于组装和交换针对真菌合成生物学定制的DNA元素。

在丝状真菌的研究和生物技术开发中，当前的挑战是DNA克隆的优化和模型真菌以外的真菌遗传转化，研究社区之间现成和标准化遗传元素的公开交换以及通用合成生物学的可用性工具和规则。GoldenBraid（GB）克隆框架是基于金门的DNA克隆系统，通过根癌农杆菌介导的遗传转化（ATMT）为植物合成生物学开发。在这项研究中，我们通过以下方法开发了用于将GB版本3.0从植物适应丝状真菌的试剂：（i）通过驯化真菌特异性遗传元件来扩展GB工具箱；（ii）真菌特异性GB结构的设计；（iii）植物病原体青霉菌的ATMT和基因破坏作为概念证明。驯化到GB进入载体pUPD2中的遗传元件包括启动子，正负选择标记和终止子。有趣的是，某些GB元素可以在植物和真菌之间直接交换，如HygR的标记hph或荧光蛋白报道基因YFP所示。元素的迭代模块化组装在pDGB3α/pDGB3Ω目的向量中产生无数个多样的转录单位和其他更高阶的组合。此外，此处对原始植物GB语法进行了修改，以纳入特定的GB结构，以通过同源重组和双重选择来破坏基因。因此，我们已经成功地将GB技术应用于真菌的ATMT。