Genetically-modified activation strategy facilitates the discovery of sesquiterpene-derived metabolites from Penicillium brasilianum

Genome mining has revealed that Penicillium spp. possess numerous down-regulated or cryptic biosynthetic gene clusters (BGCs). This finding hinted that our investigation of fungal secondary metabolomes is limited. Herein, we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by Penicillium brasilianum CGMCC 3.4402. The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene berA responsible for berkeleyacetals biosynthesis from Neosartorya glabra. Chemical analysis of the extracts from the mutant strain Pb-OE:berA enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C (1), one daucane-type arpenicarotane C (4), along with four known sesquiterpenoids including arpenibisabolane A (2), eupenicisirenins A (3), arpenicarotane B (5) and aspterric acid (6). The assignments of their structures were elucidated from detailed analyses of spectroscopic data, electronic circular dichroism calculation, and biogenetic considerations. The bioassay of isolated compounds (1-6) exhibited no cytotoxic activities against three tumor cells including MCF-7, HepG2, and A549. Arpenibisabolane C (1) and A (2) showed weak inhibition bioactivities on aquatic pathogens Vibrio owensii and Vibrio algivorus. Moreover, phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed. Based on the chemical structures and biogenetic investigations, a hypothetic pathway of new compounds (1, 4 ) was proposed.