Construction of a novel meroterpenoid biosynthetic pathway inAspergillus oryzae
Takayoshi AWAKAWA
Graduate School of Pharmaceutical Sciences, University of Tokyo
Research aims
Fungal meroterpenoids are a structurally complex class of compounds that include
pharmaceutically important molecules, such as pyripyropene A and mycophenolic acid1,2.
The study of the enzymes involved in the biosynthetic pathway of meroterpenoids will
provide a knowledge for future enzyme engineering aiming to the production of the system
to yield these compounds easily. This research focuses on sequential oxidations catalyzed bynon-heme iron dependent oxygenases2 involved in fungal meroterpenoid biosynthesis. The
goal of the project is to answer the following questions:
1. How diverse are the reactions of non-heme iron dependent oxygenases in fungal
meroterpenoid biosyntheses?
2. How do non-heme iron dependent oxygenases control their reactivity?
Once we delineate the biosynthetic reactions that produce meroterpenoids, a heterologous
expression system to produce diverse fungal meroterpenoids will be constructed.
Methods
We have previously reported two enzymes of non-heme iron dependent oxygenase family,
including AusE, an enzyme that catalyzes the addition of a spiro-lactone ring preaustinoid
A1 to produce austinol. Structural similarities between austinol and paraherquonin, derived
from the different oxidation reactions from preaustinoid A1, suggest that both compounds
are derived from preaustinoid A1. Therefore, we set out to investigate AusE homologs that
accept the same substrate, preaustinoid A1, but catalyze different types of oxidation. First,
we sequenced the genome of Penicillium brasilianum NBRC 6234, a paraherquonin
producer. The biosynthesis genes were found using a BLAST search with known
meroterpenoid terpene cyclase genes as a query. The resultant genes were expressed inAspergillus oryzae NSAR1, a fungi for a multigene expression3. The compounds that
accumulated in the transformants will be structurally determined via NMR and MS analyses.
From the structures, we will be able to deduce the reaction of each biosynthetic enzyme andto use this knowledge to construct a production system of fungal meroterpenoid compounds.

Results
From the genome sequence of P. brasilianum NBRC 6234, we searched for a genomicregion that encodes enzymes homologous to those encoded by the aus cluster and found a
possible biosynthetic gene cluster for paraherquonin, designated as the prh cluster, thatconsisted of 14 genes altogether. Of these, four genes encoding oxidative enzymes, PrhI,PrhJ, PrhK, and PrhA, were chosen from the fourteen genes, and introduced into theheterologous host Aspergillus oryzae NSAR1, and the resulting transformants werecultivated in the presence of the substrate protoaustinoid A. The transformant produced fromthe introduction of prhI alone transformed protoaustinoid A into the new metabolite
protoaustinoid B (Figure 1). The two or three gene-expression system consisting of prhI andprhJ, with or without prhK, were subsequently analyzed, with the result that thetransformants produced preaustinoid A and preaustinoid A1, respectively (Figure 1). Finally,
the strain harboring all four genes converted protoaustinoid A into berkeleydione (Figure 1).
Hence, it was demonstrated that PrhA has a different activity than AusE and that PrhA
produces berkeleydione from protoaustinoid A1 via berkeleyone B by constructing a
cycloheptadiene system. These data indicate that we successfully constructed a new system
to produce fungal meroterpenoids4.

Figure 1. The production of fungal meroterpenoids using paraherquonin biosyntheticenzymes
We proposed that the conversion of berkeleyone B to berkeleydione could occur according
to the following mechanism (Figure 2). Initially, α-KG and molecular oxygen areincorporated into the active site of the enzyme, where subsequent oxidative decarboxylationof α-KG leads to the generation of the highly active Fe(IV)–oxo species. The enzyme thenabstracts the hydrogen atom from C-1 to yield the radical species 11, which is followed by aC-C bond formation between C-1 and C-5 to give 12. The C-C bond cleavage between C-5and C-10 then achieves the expansion of the B-ring, providing 13 with a radical at the C-10
position. Finally, the abstraction of the hydrogen atom from C-1 finalizes the reaction toafford the cycloheptadiene skeleton of berkeleydione. At this point, we analyzed the enzyme
structure of PrhA via X-ray crystallization in order to compare it to that of AusE.

Figure 2. The reaction mechanism of PrhAConclusion
In this study, we identified the biosynthetic paraherquonin gene cluster and revealed themolecular pathway leading to berkeleydione. Fascinatingly, we found that the dioxygenase
PrhA catalyzes not only multistep oxidations but also a structural rearrangement to afford thecycloheptadiene moiety. To further clarify the factors that define their versatility, protein
crystallization and mutational studies of PrhA and AusE are ongoing in our laboratory. The
knowledge obtained in this research will be applied to produce unnatural fungal
meroterpenoids. We truly appreciate the “Young Investigator Research Grant from Noda
Institute for Scientific Research” that was used to fund this research.
References
1) Geris, R., Simpson, T. (2009) Meroterpenoids produced by fungi. Nat. Prod. Rep. 26:
1063–1094.
2) Matsuda, Y., Abe, I. (2016) Biosynthesis of fungal merotereonoids. Nat. Prod. Rep. 33:
26–53.
3) Jin, F. J., Maruyama, J., Juvvadi, P. R., Arioka, M., Kitamoto, K. (2004) FEMS Microbiol.
Lett. 239, 79-85.
4) Matsuda, Y. et al. (2016) Discovery of key dioxygenases that diverged the paraherquoninand acetoxydehydroaustin pathways in Penicillium brasilianum. J. Am. Chem. Soc. 138:
12671–12676.