Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

中文翻译：

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阻断药物外流机制促进了超纤维素分解真菌青霉NCIM1228的基因组工程过程

真菌青霉NCIM1228是一种非模式丝状真菌，可产生用于木质纤维素生物质糖化的高质量分泌蛋白。尽管具有理想的特性成为工业用马，但由于缺乏可靠的基因工程工具，人们一直低估了P. funiculosum。对普通真菌抗生素的耐受性一直是开发针对非模型真菌的可靠转化工具的主要障碍之一。在这项研究中，我们试图了解P. funiculosum的药物耐受性机制及其对策。然后，我们试图为该真菌的基因组工程确定一种可靠的转化方法。真菌青霉菌对潮霉素，zeocin和nourseothricin的耐药性很高，因此阻碍了它们用作获得重组转化子的选择标记。转录组分析表明，属于ABC和MFS家族的外排泵高水平表达，尤其是在生长培养基中使用复杂碳时。结合外排泵抑制剂和合适的碳源来优化抗生素选择培养基，以防止药物耐受。尝试了原生质体介导和农杆菌介导的转化以鉴定线性和环状DNA在进行基因操作中的效率。在发现基于Ti质粒的农杆菌介导的转化更适合于假单胞菌之后，我们改进了该系统，以分别实现基于随机和同源重组的基因整合和缺失。我们发现T-DNA盒的单拷贝随机整合，并且可以在基于同源重组的基因缺失中达到60％的效率。还为真菌假单胞菌开发了一种更快，无质粒且基于原生质体的CRISPR / Cas9基因编辑系统。为了显示其在P. funiculosum中的效用，我们使用一对指向cbh1开放阅读框两端的sgRNA，删除了编码最丰富的纤维素酶纤维二糖水解酶I（CBH1）的基因。Δcbh1菌株的功能分析表明，它对P. funiculosum secretome的纤维素分解特性至关重要。在这项研究中，我们解决了体育假单胞菌的药物耐受性，并开发了用于其基因组修饰的优化工具包。因此，我们为P的基因功能分析和进一步的遗传改良奠定了基础。