A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis.
A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis.
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DOI：
10.1021/acssynbio.2c00502
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发表时间：
2023-03
影响因子：
4.7
通讯作者：
Casey A. Hooker;Radwa A. Hanafy;Ethan T. Hillman;Javier Muñoz Briones;Kevin V. Solomon
中科院分区：
文献类型：
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作者：
Casey A. Hooker;Radwa A. Hanafy;Ethan T. Hillman;Javier Muñoz Briones;Kevin V. Solomon
厌氧真菌是生物技术的强大平台，由于缺乏遗传工具而尚未开发。这些肠道真菌编码真菌王国中最大数量的木质纤维素分解碳水化合物活性酶（CAZymes），使其在可再生能源和可持续性方面的应用具有吸引力。然而，遗传修饰厌氧真菌的努力仍然有限，由于DNA摄取的方法效率低下，缺乏特征性的遗传部分。我们证明，厌氧真菌是自然胜任的DNA和利用这一点来开发一个新生的遗传工具箱通知最近获得的基因组的厌氧真菌的瞬时转化。我们验证了多个选择性标记（HygR和Neo），厌氧报告蛋白（iRFP702），烯醇化酶和TEF1A启动子，TEF1A终止子，以及用于蛋白质区室化的核定位标签。这项工作建立了可靠地转化厌氧真菌Neocallimastix frontalis的新方法，从而为菌株开发和各种合成生物学应用铺平了道路。
Anaerobic fungi are powerful platforms for biotechnology that remain unexploited due to a lack of genetic tools. These gut fungi encode the largest number of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom, making them attractive for applications in renewable energy and sustainability. However, efforts to genetically modify anaerobic fungi have remained limited due to inefficient methods for DNA uptake and a lack of characterized genetic parts. We demonstrate that anaerobic fungi are naturally competent for DNA and leverage this to develop a nascent genetic toolbox informed by recently acquired genomes for transient transformation of anaerobic fungi. We validate multiple selectable markers (HygR and Neo), an anaerobic reporter protein (iRFP702), enolase and TEF1A promoters, TEF1A terminator, and a nuclear localization tag for protein compartmentalization. This work establishes novel methods to reliably transform the anaerobic fungus Neocallimastix frontalis, thereby paving the way for strain development and various synthetic biology applications.