Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, Minnesota, USA.
Department of Plant and Microbial Biology, University of Minnesota, St. Paul, Minnesota, USA.
Microbiol Spectr. 2023 Feb 14;11(1):e0424622. doi: 10.1128/spectrum.04246-22. Epub 2023 Jan 18.
Brown rot fungi are primary decomposers of wood and litter in northern forests. Relative to other microbes, these fungi have evolved distinct mechanisms that rapidly depolymerize and metabolize cellulose and hemicellulose without digesting the more recalcitrant lignin. Its efficient degradative system has therefore attracted considerable attention for the development of sustainable biomass conversion technologies. However, there has been a significant lack of genetic tools in brown rot species by which to manipulate genes for both mechanistic studies and engineering applications. To advance brown rot genetic studies, we provided a gene-reporting system that can facilitate genetic manipulations in a model fungus . We first optimized a transformation procedure in , and then transformed the fungus into a constitutive laccase producer with a well-studied white rot laccases gene (from ). With this, we built a gene reporting system based on laccase gene's expression and its rapid assay using an 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) indicator dye. The laccase reporter system was validated robust enough to allow us to test the effects of donor DNA's formats, protoplast viability, and gene regulatory elements on transformation efficiencies. Going forward, we anticipate the toolset provided in this work would expedite phenotyping studies and genetic engineering of brown rot species. One of the most ubiquitous types of decomposers in nature, brown rot fungi, has lacked robust genetic tools by which to manipulate genes and understand its biology. Brown rot fungi are primary decomposers in northern forests helping recycle the encased carbons in trees back to ecosystem. Relative to other microbes, these fungi employ distinctive mechanisms to disrupt and consume the lignified polysaccharides in wood. Its decay mechanism allows fast, selective carbohydrate catabolization, but without digesting lignin-a barren component that produces least energy trade back for fungal metabolisms. Thus, its efficient degradative system provides a great platform for developing sustainable biotechnologies for biomass conversions. However, progress has been hampered by the lack genetic tools facilitating mechanistic studies and engineering applications. Here, the laccase reporter system provides a genetic toolset for genetic manipulations in brown rot species, which we expect would advance relevant genetic studies for discovering and harnessing the unique fungal degradative mechanisms.
褐腐真菌是北方森林中木材和凋落物的主要分解者。与其他微生物相比，这些真菌已经进化出独特的机制，可以快速解聚和代谢纤维素和半纤维素，而不会消化更顽固的木质素。因此，其高效的降解系统引起了人们对开发可持续生物质转化技术的极大关注。然而，褐腐真菌缺乏遗传工具，无法对基因进行操作，无论是进行机制研究还是工程应用。为了推进褐腐菌的遗传研究，我们提供了一个基因报告系统，可以方便模型真菌中的基因操作。我们首先在 中优化了转化程序，然后用一个经过充分研究的白腐菌漆酶基因（来自 ）将真菌转化为组成型漆酶产生菌。有了这个基因报告系统，我们建立了一个基于漆酶基因表达及其利用 2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）指示剂染料的快速测定的基因报告系统。该漆酶报告系统验证结果稳健，足以让我们测试供体 DNA 格式、原生质体活力和基因调控元件对转化效率的影响。展望未来，我们预计这项工作提供的工具集将加速褐腐菌的表型研究和遗传工程。
在自然界中，最普遍的分解者之一褐腐真菌缺乏强大的遗传工具来操纵基因并了解其生物学特性。褐腐真菌是北方森林中的主要分解者，有助于将树木中包裹的碳重新循环到生态系统中。与其他微生物相比，这些真菌采用独特的机制来破坏和消耗木质素化的多糖。它的腐烂机制允许快速、选择性的碳水化合物分解代谢，但不会消化木质素——一种贫瘠的成分，为真菌代谢提供的能量交换最少。因此，其高效的降解系统为开发可持续的生物质转化生物技术提供了一个很好的平台。然而，由于缺乏促进机制研究和工程应用的遗传工具，进展受到了阻碍。在这里，漆酶报告系统为褐腐真菌的遗传操作提供了一个遗传工具集，我们预计这将推进相关的遗传研究，以发现和利用独特的真菌降解机制。
