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.