Creation of novel caspase-1 inhibitors through biosynthetic
studies of berkeleydione
Yudai MATSUDA
Graduate School of Pharmaceutical Sciences, The University of Tokyo

Research aims

Figure 1. Structures of berkeleydione (1) and its related natural products

Berkeleydione (1) is a natural product that was isolated as an inhibitor of caspase-1 and as apotential inhibitor of interleukin 1-β production by inflammasomes; it serves as the key
intermediate for the biosynthesis of many other meroterpenoids.1) Thus, biosynthetic studiesof 1 should contribute to clarifying the biosyntheses of many medicinally important naturalproducts. In this study, we aimed to determine the biosynthetic pathway of 1 at the molecularlevel, and to enzymatically derivatize 1 into a compound with improved biological activity.
Methods
Penicillium brasilianum NBRC 6234 was obtained from the National Institute ofTechnology and Evaluation. Metabolites derived from each transformant were analyzed
using HPLC. Then, prh gene-derived compounds were purified via silica-gel
chromatography, followed by preparative HPLC, and were characterized using MS andNMR analyses.
Results
To obtain biosynthetic genes for producing berkeleydione (1), we initially focused onparaherquonin, which appears to be biosynthesized via 1. We then performed whole genome
sequencing of P. brasilianum NBRC 6234, a known producer of paraherquonin, anddiscovered a putative biosynthetic gene cluster (the prh cluster) for paraherquonin. The firstfive steps of paraherquonin biosynthesis were predicted to be identical to those of austinol,producing the tetracyclic compound protoaustinoid A (2).

Figure 2. Biosynthetic pathway of berkeleydione (1)Theprhcluster

encodeseightputativetailoring

enzymes,includingone

Fe(II)/α-ketoglutarate(α-KG)-dependent
dioxygenase(PrhA),
oneshort-chain
dehydrogenase/reductase (SDR; PrhI), and two FAD-dependent monooxygenases (FMOs;PrhJ and PrhK). Since close homologs of PrhI are utilized in the biosyntheses of several
other meroterpenoids to oxidize the C-3 alcohol group of the terpene cyclase product, we
reasoned that PrhI accepts the cyclized product 2 and performs the C-3 oxidation to yield 3.
The next two conversions would involve two FMOs, PrhJ and PrhK, which are similar toAusB and AusC, respectively. Because AusB and AusC are engaged in C-5′ hydroxylation
and Baeyer-Villiger oxidation, respectively, it is most likely that PrhJ and PrhK perform thesame functions, and that both generate preaustinoid A (4) and preaustinoid A1 (5) from 3. In
the austinol pathway, 5 then undergoes two successive oxidations—catalyzed by
Fe(II)/α-KG-dependent dioxygenase AusE—to yield preaustinoid A3 with a spirolactone
system, via preaustinoid A2. The prh cluster does encode a protein homologous to AusE,PrhA; however, given the structural differences between paraherquonin and austinol, PrhA
should have an activity distinct from that of AusE. Thus, it seemed that PrhA would accept 5
but generates a different product, berkeleydione (1), probably via berkeleyone B (6) (Figure2).
To prove a biosynthetic hypothesis for berkeleydione (1), the four genes were introduced
into a heterologous host, A. oryzae NSAR1, and the resulting transformants were cultivatedin the presence of 2. The transformant with prhI alone converted 2 into 3, which wasconfirmed to be the analog of 2 with C-3 ketone functionality, and was named protoaustinoid
B. Two-gene and three-gene expression systems, [with prhI and prhJ] and [with prhI, prhJ,and prhK], respectively, were subsequently analyzed, and their transformants produced 4 and5, respectively. Finally, the strain harboring all four genes converted 2 into 1, as well as into

7. These analyses demonstrated that PrhA had an activity different from that of AusE. We
then constructed a three-gene expressing system lacking the FMO gene prhK, because theberkeleydione-producing fungus also yielded berkeleytrione (7), which seemed to besynthesized without Baeyer-Villiger oxidation. The transformant successfully produced 7,
which should be produced via 8. Collectively, PrhA is a multifunctional dioxygenase thataccepts both 4 and 5 to perform two sequential oxidations.
Conclusion
In this study, we determined the molecular basis for the biosynthesis of berkeleydione (1).2)
In future studies, we will analyze the functions of the enzymes that convert 1 into
paraherquonin, and we will construct artificial metabolic pathways using the prh genes andtheir homologs. This could contribute to the production of berkeleydione analogs withimproved biological activity.
References
1) Matsuda, Y., Abe, I. (2016) Biosynthesis of fungal meroterpenoids. Nat. Prod. Rep. 33:26–53.
2)

Matsuda, Y. et al. (2016) Discovery of key dioxygenases that diverged theparaherquonin and acetoxydehydroaustin pathways in Penicillium brasilianum. J. Am.
Chem. Soc. 138: 12671–12677.