A bacterial- like Pictet-Spenglerase drives the evolution of fungi to produce -carboline glycosides together with separate genes

Diverse beta-carboline (beta C) alkaloids are produced by microbes, plants, and animals with myriad bioactivities and drug potentials. However, the biosynthetic mechanism of beta Cs remains largely elusive, especially regarding the hydroxyl and glucosyl modifications of beta Cs. Here, we report the presence of the bacterial-like Pictet-Spenglerase gene Fcs1 in the entomopathogenic Beauveria fungi that can catalyze the biosynthesis of the beta C skeleton. The overexpression of Fcs1 in Beauveria bassiana led to the identification of six beta C methyl glycosides, termed bassicarbosides (BCSs) A-F. We verified that the cytochrome P450 (CYP) genes adjacent to Fcs1 cannot oxidize beta Cs. Alternatively, the separated CYP684B2 family gene Fcs2 was identified to catalyze beta C hydroxylation together with its cofactor gene Fcs3. The functional homologue of Fcs2 is only present in the Fcs1-containing fungi and highly similar to the Fcs1-connected yet nonfunctional CYP. Both evolved quicker than those from fungi without Fcs1 homologues. Finally, the paired methyl/glucosyl transferase genes were verified to mediate the production of BCSs from hydroxy-beta Cs. All these functionally verified genes are located on different chromosomes of Beauveria, which is in contrast to the typical content-clustered feature of fungal biosynthetic gene clusters (BGCs). We also found that the production of BCSs selectively contributed to fungal infection of different insect species. Our findings shed light on the biosynthetic mechanism of beta C glycosides, including the identification of a beta C hydroxylase. The results of this study also propose an evolving process of fungal BGC formation following the horizontal transfer of a bacterial gene to fungi.