Development of a Transposon-Based Genome Engineering Toolkit for Efficient and Adaptable Genetic Modifications in Wolfiporia cocos.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2025-04-18 Epub Date: 2025-04-02 DOI:10.1021/acssynbio.4c00766

Seungwoo Baek, Bogun Kim, Duleepa Pathiraja, In-Geol Choi

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Abstract

Advances in genome engineering of fungal strains are rapidly progressing, driven by the increasing interest in fungal biotechnology. Given the unique genomic and cellular complexity of fungi, each strain benefits from a tailored toolkit for efficient genome engineering. Here, we present a transposon-based engineering toolkit specifically optimized for Wolfiporia cocos, a species valued for its bioactive compounds. This toolkit significantly improves transformation efficiency, enabling multiplexed gene integration and facilitating rapid, flexible prototyping by assembling multiple genes into transposomes in a cocktail format, which bypasses the need for an intricate genetic circuit assembly. Engineered strains demonstrated stable expression across generations, as confirmed by successful genomic integration. Additionally, we identified six native W. cocos promoters from transcriptomic data, with two showing robust, constitutive expression in the mycelium of engineered strains. This transposon-based toolkit offers a versatile resource for synthetic biology, supporting efficient and adaptable genetic modifications within fungal systems.

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基于转座子的cocos高效适应性基因修饰基因组工程工具包的开发。

随着人们对真菌生物技术的兴趣日益浓厚，真菌菌株基因组工程的研究进展迅速。鉴于真菌独特的基因组和细胞复杂性，每个菌株都受益于高效基因组工程的量身定制工具包。在这里，我们提出了一个基于转座子的工程工具包，专门针对Wolfiporia cocos进行了优化，该物种因其生物活性化合物而受到重视。该工具包显著提高了转化效率，实现了多路基因整合，并通过将多个基因以鸡尾酒形式组装到转座体中，从而实现了快速、灵活的原型设计，从而绕过了复杂的遗传电路组装的需要。工程菌株表现出稳定的跨代表达，成功的基因组整合证实了这一点。此外，我们从转录组学数据中鉴定出6个天然W. cocos启动子，其中两个在工程菌株的菌丝体中表现出强劲的组成性表达。这个基于转座子的工具包为合成生物学提供了一个通用的资源，支持真菌系统内有效和适应性强的遗传修饰。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.