Terpenes are the largest class of natural products with extensive structural diversity and are widely used as pharmaceuticals, herbicides, flavourings, fragrances, and biofuels. While they have mostly been isolated from plants and fungi, the availability and analysis of bacterial genome sequence data indicates that bacteria also possess many putative terpene synthase genes. In this study, we further explore this potential for terpene synthase activity in bacteria. Twenty two potential class I terpene synthase genes (TSs) were selected to represent the full sequence diversity of bacterial synthase candidates and recombinantly expressed in E. coli. Terpene synthase activity was detected for 15 of these enzymes, and included mono-, sesqui-and diterpene synthase activities. A number of confirmed sesquiterpene synthases also exhibited promiscuous monoterpene synthase activity, suggesting that bacteria are potentially a richer source of monoterpene synthase activity then previously assumed. Several terpenoid products not previously detected in bacteria were identified, including aromandendrene, acora-3,7(14)-diene and longiborneol. Overall, we have identified promiscuous terpene synthases in bacteria and demonstrated that terpene synthases with substrate promiscuity are widely distributed in nature, forming a rich resource for engineering terpene biosynthetic pathways for biotechnology.

Background: Recently, a gene cluster responsible for biosynthesis of ustiloxin in Aspergillus flavus was identified as the first case of a ribosomally synthesized and post-translationally modified peptide (RiPP) synthetic pathway in Ascomycota. RiPPs are biosynthesized from precursor peptides, which are processed to produce the RiPP backbone (core peptides) for further modifications such as methylation and cyclization. Ustiloxin precursor peptide has two distinctive features: a signal peptide for translocation into the endoplasmic reticulum and highly repeated core sequences cleaved by Kex2 protease in the Golgi apparatus. On the basis of these characteristics, the ustiloxin-type RiPP precursor peptides or Kex2-processed repeat proteins (KEPs) in strains belonging to the Fungi kingdom were computationally surveyed, in order to investigate the distribution and putative functions of KEPs in fungal ecology. Results: In total, 7878 KEPs were detected in 1345 of 1461 strains belonging to 8 phyla. The average number of KEPs per strain was 5.25 in Ascomycota and 5.30 in Basidiomycota, but only 1.35 in the class Saccharomycetes (Ascomycota) and 1.00 in the class Tremellomycetes (Basidiomycota). The KEPs were classified into 838 types and 2560 standalone ones, which had no homologs. Nearly 200 types were distributed in more than one genus, and 14 types in more than one phylum. These types included yeast α-mating factors and fungal pheromones. Genes for 22% KEPs were accompanied by genes for DUF3328-domain-containing proteins, which are indispensable for cyclization of the core peptides. DUF3328-domain-containing protein genes were located at an average distance of 3.09 genes from KEP genes. Genes for almost all (with three exceptions) KEPs annotated as yeast α-mating factors or fungal pheromones were not accompanied by DUF3328-domain-containing protein genes. Conclusion: KEPs are widely distributed in the Fungi kingdom, but their repeated sequences are highly diverse. From these results and some examples, a hypothesis was raised that KEPs initially evolved as unmodified linear peptides (e.g., mating factors), and then those that adopted a modified cyclic form emerged (e.g., toxins) to utilize their strong bioactivity against predators and competitive microorganisms.

Background: The filamentous fungus Aspergillus oryzae is widely used for secondary metabolite production by heterologous expression; thus, a wide variety of promoter tools is necessary to broaden the application of this species. Here we built a procedure to survey A. flavus genes constitutively highly expressed in 83 transcriptome datasets obtained under various conditions affecting secondary metabolite production, to find promoters useful for heterologous expression of genes in A. oryzae. Results:To test the ability of the promoters of the top 6 genes to induce production of a fungal secondary metabolite, ustiloxin B, we inserted the promoters before the start codon of ustR, which encodes the transcription factor of the gene cluster responsible for ustiloxin B biosynthesis, in A. oryzae. Four of the 6 promoters induced ustiloxin B production in all tested media (solid maize, liquid V8 and PDB media), and also ustR expression. Two of the 4 promoters were those of tef1 and gpdA, which are well characterized in A. oryzae and A. nidulans, respectively, whereas the other two, those of AFLA_030930 and AFLA_113120, are newly reported here and show activities comparable to that of the gpdA promoter with respect to induction of gene expression and ustiloxin B production. Conclusion:We newly reported two sequences as promoter tools for secondary metabolite production in A. oryzae. Our results demonstrate that our simple strategy of surveying for constitutively highly expressed genes in large-scale transcriptome datasets is useful for finding promoter sequences that can be used as heterologous expression tools in A. oryzae.

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.

KK-1, a cyclic depsipeptide with 10 residues produced by a filamentous fungus Curvularia clavata BAUA-2787, is a promising pesticide active compound with high activity against many plant pathogens, especially Botrytis cinerea. As a first step toward the future mass production of KK-1 through synthetic biological approaches, we aimed to identify the genes responsible for the KK-1 biosynthesis. To achieve this, we conducted whole genome sequencing and transcriptome analysis of C. clavata BAUA-2787 to predict the KK-1 biosynthetic gene cluster. We then generated the overexpression and deletion mutants for each cluster gene using our originally developed transformation system for this fungus, and analyzed the KK-1 production and the cluster gene expression levels to confirm their involvement in KK-1 biosynthesis. As a result of these, a region of approximately 71 kb was found, containing 10 open reading frames, which were co-induced during KK-1 production, as a biosynthetic gene cluster. These include kk1B, which encodes nonribosomal peptide synthetase with a domain structure that is consistent with the structural features of KK-1, and kk1F, which encodes a transcription factor. The overexpression of kk1F increased the expression of the entire cluster genes and, consequently, improved KK-1 production, whereas its deletion decreased the expression of the entire cluster genes and almost eliminated KK-1 production, demonstrating that the protein encoded by kk1F regulates the expressions of the other nine cluster genes cooperatively as the pathway-specific transcription factor. Furthermore, the deletion of each cluster gene caused a reduction in KK-1 productivity, indicating that each gene is involved in KK-1 production. The genes kk1A, kk1D, kk1H, and kk1I, which showed a significant decrease in KK-1 productivity due to deletion, were presumed to be directly involved in KK-1 structure formation, including the biosynthesis of the constituent residues. kk1C, kk1E, kk1G, and kk1J, which maintained a certain level of KK-1 productivity despite deletion, were possibly involved in promoting or assisting KK-1 production, such as extracellular transportation and the removal of aberrant units incorporated into the peptide chain.

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Gluconobacter frateurii strain NBRC 103465 can efficiently produce glyceric acid (GA) from raw glycerol feedstock derived from biodiesel fuel production processes. Here, we report the 3.4-Mb draft genome sequence of G. frateurii NBRC 103465. The draft genome sequence can be applied to examine the enzymes and electron transport system involved in GA production.

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