Screening of secondary metabolites by genome shuffling
Masahiro OKADA
Department of Material and Life Chemistry, Kanagawa University
Research aims
In recent years, it has become feasible to artificially synthesize secondary metabolites byintroducing biosynthetic genes for natural products into microorganisms. However, noeffective method has yet been established to create new secondary metabolites by modifying
biosynthetic gene clusters. In order to achieve such modifications, it is necessary to gain a
deep understanding of biosynthetic gene clusters, but their fundamental genetic associations
are still poorly understood. In this work, natural products were biosynthesized to artificially
activate biosynthetic gene clusters, and then, in order to create various non-natural products,
we attempted to randomly combine gene groups by genome shuffling.
Methods and results
We selected sesterterpenoids as targets for the artificial activation of biosynthetic geneclusters. Terpenes are natural products derived from C5 isoprene units. They include a huge
number of compounds, with many different backbone skeletons, that have been used in thedevelopment of important pharmaceuticals. Sesterterpenoids are a group of terpenoid naturalproducts that are primarily biosynthesized via cyclization of the C25 linear substrate. There arerelatively rare reports on the isolation of sesterterpenoids. However, various organisms suchas bacteria, fungi, and plants produce sesterterpenoids as well as other terpenoids. Therefore,
in order to obtain sesterterpenoids, several putative terpene synthases were heterologously
expressed in Aspergillus oryzae NSAR1, as this strain is suitable for the characterization offungal biosynthetic enzymes. The genes selected and introduced in this study were identified
by the genome analysis of two Penicillium species, P. brasilianum NBRC 6234 and P.
verruculosum, which harbor putative terpene synthases.
Transformants were cultivated in induction medium and the resulting mycelial metabolites
were analyzed by high resolution mass spectrometry and nuclear magnetic resonance. As aresult, we identified two new sesterterpenes: preasperterpenoid A, derived from P.
verruculosum preasperterpenoid A synthase (PvPS) and sesterbrasiliatriene, derived from P.
brasilianum sesterbrasiliatriene synthase (PbSS) (Figure 1). 1) In addition, copalyl diphosphate
was also obtained from P. verruculosum copalyl diphosphate synthase (PvCPS) (Figure 1). 2)

Meanwhile, an oligopeptide possessing an isoprenylated tryptophan residue, ComXnatto
pheromone, was also obtained by means of heterologous overexpression (Figure 2). 3)

Figure 1. Chemical structures of sesterterpenes and diterpene (copalol).
Next, we attempted to randomly combine gene groups by genome shuffling. As a model
study, we conducted a polyethylene glycol (PEG)-mediated cell fusion between aquiannulatene 4)-overexpressing A. oryzae strain and an auxotrophic A. oryzae strain (ΔpyrGor A. ΔadeB). As a result, we obtained 5 fusion cells, but no new products were detected fromthe resulting mycelial metabolites thus far. A further cell fusion study was conducted by using
P. islandicum Sopp NBRC 6964 and P. rugulosum Thom NBRC 696. As a result, we obtained
five phenotypically distinct fusion cells. We are currently analyzing their mycelial metabolites.
In addition, the heterologous expression of enzymes derived from Rhododendron dauricum
plants and Stachybotrys bisbyi fungi mediated the three-step combinatorial biosynthesis of(+)-daurichromenic acid, which is a meroterpenoid with highly potent anti-HIV activity fromR. dauricum. Additional introduction of a halogenase from Fusarium sp. fungi into the strain
resulted in the biosynthesis of the non-natural product (+)-5-chlorodaurichromenic acid, which
exceeds the antibacterial activity of the original compounds (Figure 2). 5)
Conclusion
Heterologous expression of putative terpene synthases derived from a filamentous fungus
enabled the artificial synthesis of several novel secondary sesterterpenes and diterpenes.
Furthermore, cell fusion was successfully carried out using the strains. Unfortunately, newsecondary metabolites were not obtained from the fusion cells, but other fusion cells wereobtained with distinct phenotypes.

Figure 2. Chemical structures of the ComXnatto pheromone and (+)-5chlorodaurichromenic acid.
Acknowledgments
This work was supported in part by the Noda Institute for Scientific Research GRANT. We
deeply appreciate the financial support.
References
1) Mitsuhashi, T., Okada, M., and Abe, I. (2017) Identification of chimeric αβγ diterpene
synthases possessing both type II terpene cyclase and prenyltransferase activities.
ChemBioChem 18: 2104–2109.
2) Mitsuhashi, T., Rinkel, J., Okada, M., Abe, I., and Dickschat, J. S. (2017) Mechanistic
characterization of two chimeric sesterterpene synthases from Penicillium. Chem. Eur. J.
23: 10053–10057.
3) Sugita, T., Okada, M., Nakashima, Y., Tian, T., and Abe, I. (2018) A tryptophan
prenyltransferase with broad substrate tolerance from Bacillus subtilis subsp. natto.
ChemBioChem, 19: 1396–1399.
4) Okada, T., Matsuda, Y., Mitsuhashi, T., Hoshino, S., Mori, T., Nakagawa, K., Quan, Z.,Qin, B., Zhang, H., Hayashi, F., Kawaide, H., and Abe, I. (2016) Genome-based
discovery of an unprecedented cyclization mode in fungal sesterterpenoids biosynthesis.
J. Am. Chem. Soc. 138: 10011–10018.
5) Okada, M., Saito, K., Wong, C. P., Li, C., Wang, D., Iijima, Taura, F., Kurosaki, F.,
Awakawa, T., and Abe, I. (2017) Combinatorial biosynthesis of (+)-daurichromenic acidand its halogenated analogue. Org. Lett. 19: 3183–3186.