Analysis of FR901379 Biosynthetic Genes in <i>Coleophoma empetri</i> by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation

Abstract

The compound FR901379, a sulfated echinocandin produced by the filamentous fungus <i>Coleophoma empetri</i> 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 <i>C. empetri</i> 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 (<i>CEnrps</i>), the fatty-acyl-AMP ligase gene (<i>CEligase</i>) responsible for the formation of the activated form of palmitic acid and its transfer to <i>CEnrps</i>, four nonheme mononuclear iron oxygenase genes (<i>CEoxy1</i>, <i>CEoxy2</i>, <i>CEoxy3</i>, and <i>CEoxy4</i>) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (<i>CEhtyA-D</i>), two cytochrome P450 enzyme genes (<i>CEp450-1</i> and <i>CEp450-2</i>), and a transcription regulator gene (<i>CEhyp</i>). In addition, by screening the whole genome, we identified two unknown genes (<i>CEp450-3</i> and <i>CEsul</i>) 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.