Search for transcription factors affecting productivity of the polyketide FR901512 in filamentous fungal sp. No. 14919 and identification of Drf1, a novel negative regulator of the biosynthetic gene cluster.

In order to increase secondary metabolite production in filamentous fungi, a transcription factor gene in the biosynthetic gene cluster and global regulator genes such as laeA are considered plausible as targets for overexpression by genetic modification. In this study, we examined these overexpression effect in fungal sp. No. 14919 that produces FR901512, an HMG-CoA reductase inhibitor. Resultantly, the productivity was improved at 1.7-1.8 fold by overexpressing frlE, a transcription factor gene in the biosynthetic gene cluster, whereas productivity did not change by overexpression of laeA and veA. Furthermore, we searched for extra transcription factors affecting the productivity by transcriptome analysis between wild-type strain and highly productive UV mutants. After verifying productivity decrease by overexpression, Drf1, a novel transcription factor encoded by drf1 was identified as the negative regulator. Because each frlE product (FrlE) and Drf1 worked on the same cluster in positive and negative regulatory manners, their network was analyzed.

Biosynthesis of Novel Statins by Combining Heterologous Genes from Xylaria and Aspergillus.

For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes. Key structure determinants of statins are essential accessory genes that are irreplaceable across species.

Motif-independent prediction of a secondary metabolism gene cluster using comparative genomics: application to sequenced genomes of Aspergillus and ten other filamentous fungal species.

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters.

Genome Sequence of the Mucoromycotina Fungus Umbelopsis isabellina, an Effective Producer of Lipids.

Umbelopsis isabellina is a fungus in the subdivision Mucoromycotina, many members of which have been shown to be oleaginous and have become important organisms for producing oil because of their high level of intracellular lipid accumulation from various feedstocks. The genome sequence of U. isabellina NBRC 7884 was determined and annotated, and this information might provide insights into the oleaginous properties of this fungus.

Fine de novo sequencing of a fungal genome using only SOLiD short read data: verification on Aspergillus oryzae RIB40.

The development of next-generation sequencing (NGS) technologies has dramatically increased the throughput, speed, and efficiency of genome sequencing. The short read data generated from NGS platforms, such as SOLiD and Illumina, are quite useful for mapping analysis. However, the SOLiD read data with lengths of <60 bp have been considered to be too short for de novo genome sequencing. Here, to investigate whether de novo sequencing of fungal genomes is possible using only SOLiD short read sequence data, we performed de novo assembly of the Aspergillus oryzae RIB40 genome using only SOLiD read data of 50 bp generated from mate-paired libraries with 2.8- or 1.9-kb insert sizes. The assembled scaffolds showed an N50 value of 1.6 Mb, a 22-fold increase than those obtained using only SOLiD short read in other published reports. In addition, almost 99% of the reference genome was accurately aligned by the assembled scaffold fragments in long lengths. The sequences of secondary metabolite biosynthetic genes and clusters, whose products are of considerable interest in fungal studies due to their potential medicinal, agricultural, and cosmetic properties, were also highly reconstructed in the assembled scaffolds. Based on these findings, we concluded that de novo genome sequencing using only SOLiD short reads is feasible and practical for molecular biological study of fungi. We also investigated the effect of filtering low quality data, library insert size, and k-mer size on the assembly performance, and recommend for the assembly use of mild filtered read data where the N50 was not so degraded and the library has an insert size of ∼2.0 kb, and k-mer size 33.