Construction of biosynthetic machinery for polyketides witha bis-tetrahydrofuran moiety
Makoto HASHIMOTO
School of Pharmacy, Iwate Medical University
Research aims
Natural products containing a bis-tetrahydrofuran moiety are reported to have attractive
biological activities.1, 2) For example, asteltoxin (1), a polyketide found in Emericellavariecolor, is a potent ATPase inhibitor. In its production, a hexaene-fused α-pyrone (2) isformed by polyketide synthase (PKS), followed by epoxidation and hydration steps that
result in a bis-tetrahydrofuran formation (Fig. 1).3) The regio-selective bis-tetrahydrofuran
formation and potential biological activity prompted us to investigate the asteltoxin
biosynthetic machinery, which could lead to the preparation of asteltoxin derivatives as drugcandidates. The draft genome analysis of E. variecolor IFM42010 found two candidate
asteltoxin gene clusters, namely Ev460 and Ev152 (Fig. 2A, 3A). We previously determinedthat Ev460PKS produces a hexaene-fused β-keto lactone (4), whose side-chain is the sameas that of (2) (Fig. 2B). Herein, we attempted the identification of the gene cluster
responsible for the biosynthesis of asteltoxin based on gene disruption and biochemical
analysis of the initial step of asteltoxin biosynthesis.
Methods
The Agrobacterium tumefaciens-mediated transformation (ATMT) method was utilized togenerate E. variecolor IFM42010 knockout mutants. The disruption cassette containing hph
flanked by 2 kb of Ev460PKS gene at each end were cloned into the T-DNA region of
pRI201AN to construct the disruption plasmid pRI201AN-hph-Ev460PKS. Agrobacteria
carrying the disruption plasmid were incubated with conidia of E. variecolor on a nylon
filter placed on a co-cultivation plate with acetosyringone. The transformant of E. variecolor
was then selected using a PDA plate containing hygromycin and cefotaxime.
Aspergillus oryzae NSAR1, a quadruple auxotrophic mutant, was transformed by theprotoplast-PEG method with a fungal expression plasmid harboring an α-amylase promotorsystem.
Results

1

Disruption of the genes in the Ev460 cluster
The hygromycin-resistant transformants were analyzed via PCR to confirm whether

Ev460 genes were replaced with a hph cassette. To date, only one disruptant per 45 candidate
transformants was obtained using the ATMT method. HPLC analysis of the ethyl acetate
extract from the Ev460PKS disruptant revealed that Ev460PKS was not required for theasteltoxin production (Fig. 2C). Furthermore, the ∆Ev460TF2 mutant maintained asteltoxin
production. We therefore concluded that the Ev460 gene cluster is not involved in asteltoxin
biosynthesis.
2

Heterologous expression of Ev152PKSGenes in the Ev152 cluster are similar to those in the aurovertin biosynthetic genecluster.4）When the A. oryzae transformant with pTA-Ev152PKS was cultured in rice medium,a new peak was detected which was absent in the control transformant (A. oryzae
transformant with pTAex3) in the ethyl acetate extract after HPLC analysis (Fig. 3B).
LC-MS analysis of the extract suggested that the molecular formula of the new product(C22H26O3) was the same as that of the hexaene-fused α-pyrone (2) precursor to asteltoxin(Fig. 3C). Currently, co-expression experiments of other genes in the Ev152 cluster withEv152PKS are underway.
Conclusion
In this study, we attempted to identify the gene cluster responsible for asteltoxin
biosynthesis and to construct biosynthetic machinery for polyketides with abis-tetrahydrofuran moiety. A gene disruption experiment showed that the Ev460 cluster was
not involved in asteltoxin biosynthesis. Heterologous expression of Ev152PKS suggested
that the Ev152 cluster, which is similar to that of aurovertin, could be the actual asteltoxin
biosynthetic gene cluster. Further functional analyses of the Ev152 gene cluster (Fig. 3A)
could provide useful information for the reconstitution of biosynthetic machinery to produce
a bis-tetrahydrofuran moiety in polyketides.

References
1) Alonso, F., Sanchez, D., Soler, T., and Yus, M. (2008) Straightforward synthesis ofperhydrofuro [2, 3-b] furans through a Wacker-type reaction. Adv. Synth. Cat. 350:
2118–2126.
2) Sridhar, P. R., Reddy, G. M., and Seshadri, K. (2012) Stereoselective synthesis of cis-fused
perhydrofuro [2, 3-b] furan derivatives from sugar-derived allyl vinyl ethers. Eur. J. Org.
Chem. 2012: 6228–6235.

3) Eom, K. D., Raman, J. V., Kim, H., and Cha, J. K. (2003) Total synthesis of (+)-asteltoxin.
J. Ame. Chem. Soc. 125: 5415–5421.
4）Mao, X. M. et al. (2015) Efficient biosynthesis of fungal polyketides containing thedioxabicyclo-octane ring system. J. Am. Chem. Soc. 137: 11904–11907.

Fig. 1.

Putative biosynthetic pathway of asteltoxin

Fig. 2. The 460 gene cluster and HPLC analysis of Ev460PKS disruptant
A) Ev460 cluster in E. variecolor IFM42010 B) Ev460PKS product C) HPLC analysis ofethyl extract from ∆Ev460 transformantB)

A)
polyketide synthase MeT hydorolase
cyclase(PKS)
FMO
orf1

orf2 orf3 orf4 orf5(16 kb)

C)Fig. 3. The Ev152 gene cluster and HPLC analysis of A. oryzae with Ev152PKSA) Ev152 cluster in E. variecolor IFM42010 B) HPLC analysis of A. oryzae
NSAR1/pTA-Ev152PKS C) Mass chromatogram with m/z 339.1955 of the extract