42

Noda Institute for Scientific Research GRANT
2012 Young Investigator Research Grant

Screening for small molecules that activate fungal secondary
metabolite production
Naoki Kato
RIKEN Center for Sustainable Resource ScienceResearch aims
Recent progress in fungal genome sequencing indicatesfungi have a greater potential to biosynthesize secondary
metabolites (SMs) than that speculated based on number ofSMs isolated so far. Genes involved in SM biosynthesis infilamentous fungi are clustered in their genome, and theirregulation is coordinated by complex regulatory systems
consisting of global regulators and pathway-specific regulators.1) Pathway-specific regulators, including C6-type transcription factors, have been considered as valid starting
points for studying cryptic SM gene clusters.
The 120-kb subtelomeric region of chromosome 8p inAspergillus fumigatus is responsible for three biosynthetic
pathways of bioactive SMs, fumitremorgins,2,3) fumagillin,4) and pseurotin A5) (Fig. 1). While more than 60% of
the genes found in this region were predicted to be involved
in SM biosynthesis, there is only one regulatory gene,
AFUA_8G00420, in this region. However, it is unclear
whether AFUA_8G00420 regulates the biosynthesis of fumagillin or other SMs such as pseurotins and fumitremorgins. In this study, we studied the roles ofAFUA_8G00420 in SM production in A. fumigatus, and weconstructed a reporter system to screen small moleculesthat activate fungal SM production.
Methods
To examine the roles of AFUA_8G00420 in SM production in A. fumigatus, a knockout mutant (∆420) was generated by replacing the entire coding region of the gene witha hygromycin B-resistance gene cassette. The fungal strains
were cultivated in complete (0.5% yeast extract, 0.5% malt
extract, 1% glucose, and 1% soluble starch) or glucose
minimum medium at 28°C for 24 and 48 h to analyze theirgene expression and metabolite production profiles. Expression analysis was carried out by RT-PCR. Ethyl acetate
extracts of fungal cultures were analyzed by LC/MS.
Results
We compared SM gene expression in ∆420 mutant with
its parental wild-type strain. Expression of some of the fma
genes such as fmaAT and AFUA_8G00500 was lower in∆420 mutant, apparently because AFUA_8G00420 was included in the fumagillin biosynthetic gene (fma) cluster. In-Fig. 1. Biosynthetic gene clusters for fumitremorgins, pseurotins, and fumagillin, located at the 120-kb region of chromosome 8p of A. fumigatus, and apathway-specific regulator that controls the production of fumagillin and pseurotin.

		

terestingly, the mutant also showed lower expression ofsome pso genes than did the wild-type strain. We found nosignificant difference in ftm gene expression between thewild-type and ∆420 mutant strains. To determine whether∆420 mutant strain produces fumitremorgins but not fumagillin and pseurotins, metabolite profiles of the A. fumigatus
mutants were compared with those of the wild-type strains.
The deletion of AFUA_8G00420 caused a loss of production of fumagillin and pseurotin A, but not fumitremorgins,
clearly demonstrating that AFUA_8G00420 is involved in
the regulation of the biosynthetic pathways of fumagillin
and pseurotin A. These results agreed well with the expression profiling results of ∆420 mutant.
Based on the results described above, a reporter system
for the small molecule screen was constructed by fusing areporter gene to the promoter regions of AFUA_8G00420dependent, genes fmaAT and AFUA_8G00530.
Conclusion
Gene expression and metabolite production profiling of
the knockout mutant of AFUA_8G00420 demonstrated thatthe gene plays a role in the regulation of fumagillin andpseurotins, but is unlikely to be involved in fumitremorgin

43

production in A. fumigatus. To the best of our knowledge,
this is the first report of a C6-type transcriptional activator
that regulates two discrete SM biosynthetic pathways in afilamentous fungus. Better understanding of the underlying
mechanism of the unique crosstalk will allow us to exploit
cryptic SM gene clusters through this family of transcription factors. Currently, we are employing this regulatory
system and developing a screening system for small molecules that control fungal SM production.
References
1.

Brakhage, A. A. (2013) Regulation of fungal secondary metabolism.
Nat Rev Microbiol 11: 21–32.
2. Li, S. M. (2011) Genome mining and biosynthesis of fumitremorgintype alkaloids in ascomycetes. J Antibiot (Tokyo) 64: 45–49.
3. Kato, N., et al. (2009) Identification of cytochrome P450s required
for fumitremorgin biosynthesis in Aspergillus fumigatus. Chembiochem 10: 920–928.
4. Lin, H. C., Chooi, Y. H., Dhingra, S., Xu, W., Calvo, A. M., andTang, Y. (2013) The fumagillin biosynthetic gene cluster in Aspergillus fumigatus encodes a cryptic terpene cyclase involved in the formation of β-trans-bergamotene. J Am Chem Soc 135: 4616–4619.
5. Maiya, S., Grundmann, A., Li, X., Li, S. M., and Turner, G. (2007)
Identification of a hybrid PKS/NRPS required for pseurotin A biosynthesis in the human pathogen Aspergillus fumigatus. Chembiochem 8: 1736–1743.