Production of new biologically active compounds by a combined
culture of fungi and investigation of their activation mechanism
Sachiko TSUKAMOTO
Graduate School of Pharmaceutical Sciences, Kumamoto University
Research aims
Secondary metabolites produced by microorganisms have been widely used in medicine todate. Recent techniques in genome analysis revealed that most genes coding for secondary
metabolites are not expressed and that fungal cultures can provide only a few of thosemetabolites. Therefore, a variety of techniques to activate dormant genes has been developed.
We have been searching for drug candidates from marine organisms using a bioassay
screening and have identified new compounds with unique structures. We have also isolated
3000 fungi from sediments, marine organisms, and plants. The activation of dormant genes
may lead to the identification of unprecedented metabolites. In this study, we aimed to obtainnew metabolites from a combined culture of fungi and to study their mechanism ofproduction. Our results may significantly extend the metabolite profile of fungi.
Methods
Our experiments showed that fungi produce more metabolites in solid cultures than inliquid cultures. To obtain new metabolites we conducted a combined culture of fungi on agarplates.
(1) Screening of a combined culture of fungi.
Two fungi species with various combinations were cultured on malt agar plates and thecontact sections were extracted by MeOH. The MeOH extracts of the combined culture andrespective pure cultures were analyzed by LC-MS.
(2) Isolation of new metabolites from a combined culture of fungiTwo fungi species, Phaeosphaeria oryzae (13S070) and Irpex lacteus (13S052) isolatedfrom plants collected in the Aso area, were cultured on malt agar plates in large scale and theMeOH extracts were purified to acquire new compounds.
Results
The combined culture of Phaeosphaeria oryzae (13S070) and Irpex lacteus (13S052) onmalt agar plates produced a green pigment on the contact section (Figure 1). Analysis of thecontact section MeOH extract by LC-MS indicated the presence of new metabolites
generated by the combined culture, in addition to the pigment (Figure 2). Purification of thecombined culture in a large scale followed by spectroscopic analysis led to the identificationof the pigment displayed in peak 1 (Figure 2) as hypoxyxylerone1)（Figure 3）. LC-MS
analysis showed the presence of several analogs of hypoxyxylerone. Neither P. oryzae norI. lacteus produced hypoxyxylerone on malt agar medium, but I. lacteus produced it on rice
medium, which clearly indicated that the hypoxyxylerone-producing fungus was I. lacteus.
These results also revealed that the production of hypoxyxylerone by I. lacteus in malt agar
medium was induced by the contact with P. oryzae or by compounds secreted from P. oryzae.
The structure of the compound designated as peak 2 was studied by spectroscopy and foundto be a new compound.2)

Figure 1. Cultures of Phaeosphaeria oryzae (13S070) (left) and Irpex lacteus (13S052)(right) and the combined culture (middle). In the combined culture, the two fungi cultures
were inoculated 1 cm away from each other.

Figure 2. HPLC chromatogram of the culture extracts of P. oryzae, I. lacteus, and their
combined culture detected at 280 nm.

Figure 3. Structure of hypoxyxylerone.
Conclusion
Recently, pharmaceutical companies have shifted their target for drug development from
infections to chronic diseases and have withdrawn from the search for new drugs fromnatural sources. As a result, the number of new anti-infective agents in the market hasdecreased. However, the development of anti-infective agents has been conducted vigorously
all over the world, and in order to discover new anti-infective agents, the use of new
resources and the activation of dormant genes are admittedly important. So far, activation offungal genes by co-culturing them with bacteria such as actinomyces has been performed,
but co-cultures of two fungi have been hardly conducted. In this study, we showed that thefungus I. lacteus produced a new compound in a combined culture with another fungus onsolid medium. Production of new compounds from dormant genes may result in thedevelopment of new anti-infective agents, with potential applications on the fermentationand food industries as well.
References
1) Edwards, R. L., Fawcett, V., Maitland, D. J., Nettleton, R., Shields, L., and Whalley, A. J.
S. (1991) Hypoxyxylerone. A novel green pigment from the fungus Hypoxylon
fragiforme (Pers.: Fries) Kickx. J. Chem. Soc., Chem. Commun. 1009-1010.
2) Sadahiro, Y., Kato, H., Tsukamoto, S. (2018) A new metabolite produced by co-cultureof fungi, Phaeosphaeria oryzae and Irpex lacteus. In preparation.