The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

中文翻译：

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通过成簇规则间隔的短回文重复序列/基于 Cas9 的基因组操作分析 Coleophoma empetri 中的 FR901379 生物合成基因

化合物 FR901379 是由丝状真菌Coleophoma empetri F-11899 产生的硫酸化棘白菌素，是合成抗真菌药物米卡芬净的重要中间体。在这项研究中，我们为工业生产菌株C. empetri SIPI1284 建立了一种高效的成簇规则间隔短回文重复序列/基于 Cas9 的基因编辑工具。利用该方法，目标位点的基因诱变效率高达84%，可实现FR901379生物合成基因分析的快速基因破坏。接下来，我们通过体内基因破坏和基因互补验证了 FR901379 生物合成基因簇的推定功能基因。这些核心功能基因包括非核糖体肽合成酶基因（CEnrps），脂肪酰基-AMP 连接酶基因（CEligase）负责形成棕榈酸的活化形式并将其转移到CEnrps，四种非血红素单核铁加氧酶基因（CEoxy1、CEoxy2、CEoxy3和CEoxy4）负责非蛋白氨基酸的合成、l-高酪氨酸生物合成基因 ( CEhtyA-D )、两个细胞色素 P450 酶基因（CEp450-1和CEp450-2）和一个转录调节基因 ( CEhyp )。此外，通过全基因组筛选，我们发现了两个未知基因（CEp450-3和CEsul ) 负责 FR901379 的磺酰氧基，它们与核心 FR901379 生物合成簇分离。此外，在研究中的基因破坏过程中，我们获得了一系列 FR901379 类似物，并阐明了类群与抗真菌活性之间的关系。