Discovery of cryptic fungal natural products using genome
rearrangement
Teigo ASAI
Graduate School of Pharmaceutical Sciences, Tohoku University

Research objective
Filamentous fungi are important microbial resources that have contributed to thedevelopment of numerous pharmaceuticals including penicillin and lovastatin. The genome offilamentous fungi contains a large number of unused biosynthetic genes, many of which are
known to be silent genes whose expression is not sufficiently induced under normal culture
conditions to produce substances. These silent genes are expected to be valuable troves ofnovel natural products that can be used as seeds for new drug development, and methods toactivate silent genes are being actively pursued. We established a method to induce theexpression of silent genes by artificially modifying epigenetic regulation using small-moleculecompounds and have discovered novel natural products. However, the methods developed thus
far can only induce the production of natural products derived from one or two silent
biosynthetic gene clusters; these gene resources have not yet been fully exploited. In this study,
we applied the TAQing system, a large-scale genome reorganization technique, to filamentous
fungi and developed a method for activating diverse silent genes in a single species.
Methods
The TAQing system is a method of introducing the restriction enzyme TaqI (activated athigh temperature) from a highly thermophilic bacterium into the cell and causing a transient
temperature shift, which simultaneously causes multiple double-strand breaks in the genomic
DNA, followed by recombination repair, resulting in various genomic reorganizations such as
end joining, homologous recombination, translocation, and copy number variation.1 In
filamentous fungi, many secondary metabolite biosynthetic gene clusters are located near thetelomeres at the chromosome ends, and the location of these biosynthetic gene clusters plays
a major role in their expression. In this study, we established an optimal protocol using
Aspergillus niger, a model filamentous fungus for secondary metabolism studies. A plasmid
vector was created by introducing taqI downstream of the amyB and enoA promoters.
Pyrithiamine resistance was used to select the transformants. This plasmid was introduced into

A. niger using the protoplast-PEG method to obtain a taqI-expressing strain. As the introduced
TaqI shows high activity at high temperatures, we examined various heat shock conditions forthe transformants and confirmed the appearance of transformed strains by repeated subculture
at temperatures between 30°C and 37°C, where the restriction enzyme activity of TaqI ismoderately observed. Morphologically altered strains were then observed under these
conditions. These morphologically altered strains were isolated, the production profiles ofsecondary metabolites were investigated, and genomic analysis was performed.
Results
Through repeated subculture of the transformants with taqI at 30°C to 37°C, we succeeded in
obtaining diverse morphologically altered strains (Fig. 1). There was no significant differencein the expression frequency of the morphologically altered strains between the amyB and enoA
promoters, suggesting that a strong promoter that induces constant expression is sufficient forthis method. Most of the morphologically altered strains were found to have reduced
sporulation ability and mycelial growth compared to those of the parental strain; however, as
this was detected by visual morphological changes, obtaining different transformed strains
may be possible using other selection methods.

Conclusion
Fig. 1. Examples of morphologically altered strains isolated in this study
The production profiles of the secondary metabolites of the morphologically altered strainsobtained in this study were examined by HPLC after cultivation, and the results showed thatthe secondary metabolic profiles were different from those of the parental strains. The
metabolic profiles differed among the morphologically altered strains, indicating that thisstudy successfully activated diverse silent genes as intended (Fig. 2). In addition, decoding of

the genomes of the morphologically altered strains revealed large-scale gene deletions andtranslocations near the TaqI recognition site, suggesting that genome reorganization occurredowing to gene cleavage by TaqI.

Fig. 2 HPLC profiles of wild type (parental strain) and some morphologically altered strains
In summary, this study showed that heterologous expression of TaqI in filamentous fungiand subculture at 30°C to 37°C can provide morphologically altered strains. However, thedifficulty in maintaining traits and improving selection methods to identify more diverse traits
are issues for future research. Currently, we are continuing our research to solve thesechallenges and expanding our research to various other filamentous fungi to obtain diverse
new natural products.
References
1. Muramoto, N., Oda, A., Tanaka, H., Nakamura, T., Kugou, K., Suda, K., Kobayashi, A.,
Yoneda, S., Ikeuchi, A., Sugimoto, H., Kondo, S., Ohto, C., Shibata, T., Mitsukawa, N.,Ohta, K. Phenotypic diversification by enhanced genome restructuring after induction ofmultiple DNA double strand breaks. Nature Commun. 9:1995. doi: 10.1038/s41467-01804256-y. (2018).