Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of Aspergillus section Fumigati (A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae, and Neosartorya fischeri). For expanding our knowledge, we newly sequenced genomes of A. viridinutans and A. pseudoviridinutans, and reassembled and reannotated the previously released genomes of A. lentulus and A. udagawae. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7–51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using A. fumigatus, A. lentulus, A. udagawae, A. viridinutans, and N. fischeri was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32–83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

Species of the Aspergillus section Nigri are taxonomically very complex. The taxonomic assignment of Aspergillus awamori is unclear.

33Filamentous fungi produce various bioactive compounds that are biosynthesized by a 34 set of proteins encoded in biosynthetic gene clusters (BGCs). For an unknown reason, 35 large parts of the BGCs are transcriptionally silent under laboratory conditions, which 36 has hampered the discovery of novel fungal compounds. The transcriptional regulation 37 of fungal secondary metabolism is not fully understood from an evolutionary viewpoint. 38To address this issue, we conducted comparative genomic and transcriptomic analyses 39 using five closely related species of the Aspergillus section Fumigati: Aspergillus 40 fumigatus, Aspergillus lentulus, Aspergillus udagawae, Aspergillus pseudoviridinutans, 41and Neosartorya fischeri. From their genomes, 298 secondary metabolite (SM) core 42 genes were identified, with 27.4% to 41.5% being unique to a species. Compared with 43 the species-specific genes, a set of section-conserved SM core genes was expressed 44 at a higher rate and greater magnitude, suggesting that their expression tendency is 45 correlated with the BGC distribution pattern. However, the section-conserved BGCs 46 showed diverse expression patterns across the Fumigati species. Thus, not all common 47BGCs across species appear to be regulated in an identical manner. A consensus motif 48 was sought in the promoter region of each gene in the 15 section-conserved BGCs 49 among the Fumigati species. A conserved motif was detected in only two BGCs including 50 the gli cluster. The comparative transcriptomic and in silico analyses provided insights 51 into how the fungal SM gene cluster diversified at a transcriptional level, in addition to 52 genomic rearrangements and cluster gains and losses. This information increases our 53 understanding of the evolutionary processes associated with fungal secondary 54 metabolism. 55 56 KEY WORDS: comparative genomics, comparative transcriptomics, secondary 57 metabolism, biosynthetic gene cluster, Aspergillus 58 59 Author summary 61 Filamentous fungi provide a wide variety of bioactive compounds that contribute to public 62 health. The ability of filamentous fungi to produce bioactive compounds has been 63 underestimated, and fungal resources can be developed into new drugs. However, most 64 biosynthetic genes encoding bioactive compounds are not expressed under laboratory 65 conditions, which hampers the use of fungi in drug discovery. The mechanisms 66 underlying silent metabolite production are poorly understood. Here, we attempted to 67 show the diversity in fungal transcriptional regulation from an evolutionary viewpoint. To 68 meet this goal, the secondary metabolisms, at genomic and transcriptomic levels, of the 69 most phylogenetically closely related species in Aspergillus section Fumigati were 70 compared. The conserved biosynthetic gene clusters across five Aspergillus species 71were identified. The expression levels of the well-conserved gene clusters tended to be 72 more active than the species-specific, which were not well-conserved, gene clusters. 73Despite hig...