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

Genome mining has revealed that <i>Penicillium</i> 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 <i>Penicillium brasilianum</i> CGMCC 3.4402. The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene <i>berA</i> responsible for berkeleyacetals biosynthesis from <i>Neosartorya glabra</i>. Chemical analysis of the extracts from the mutant strain <i>Pb</i>-OE:<i>berA</i> enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C (<b>1</b>), one daucane-type arpenicarotane C (<b>4</b>), along with four known sesquiterpenoids including arpenibisabolane A (<b>2</b>), eupenicisirenins A (<b>3</b>), arpenicarotane B (<b>5</b>) and aspterric acid (<b>6</b>). 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 (<b>1</b>-<b>6</b>) exhibited no cytotoxic activities against three tumor cells including MCF-7, HepG2, and A549. Arpenibisabolane C (<b>1</b>) and A (<b>2</b>) showed weak inhibition bioactivities on aquatic pathogens <i>Vibrio owensii</i> and <i>Vibrio algivorus</i>. 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 (<b>1</b>, <b>4</b>) was proposed.