Terretonin Biosynthesis Requires Methylation as Essential Step for Cyclization

The fungal meroterpenoids have tremendous structural diversity. This group includes several medicinally important compounds, such as the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, pyripyropene A, the selective acetylcholinesterase inhibitor, arisugacin A, and the protein farnesyltransferase inhibitor, andrastin A (Scheme 1). Many of these compounds have polyketide and terpenoid origins. The diversities of these two groups contribute to the structural diversity of fungal meroterpenoids. Understanding the biosynthetic routes of meroterpenoids is important for utilizing the pathways for combinatorial biosynthesis. We have previously reported the functions of the enzymes catalyzing pyripyropene A biosynthesis, consisting of CoA-ligase, polyketide synthase (PKS), prenyltransferase (PT), flavin-dependent monooxygenase (FMO) and terpene cyclase (CYC). In the biosynthesis of pyripyropene A, the terpenoid moiety is cyclized by Pyr4, a novel transmembrane protein with very low similarity to the known terpene cyclases. The cyclization reaction in fungal meroterpenoid biosynthesis is one of the key steps that generate the structural diversity of this class of compounds. As exemplified by terretonin, austinol, andrastin A and anditomin, some compounds are derived from the same polyketide core and differently cyclized terpenoid moieties. These compounds are all derived from 3,5-dimethylorsellinic acid (DMOA, 1, Scheme 2), but they have various cyclic terpenoid moieties. These differences are due to the presence of CYCs with diverse cyclization activities. Terretonin is a toxic compound isolated from Aspergillus terreus. 8] The intriguing structure of terretonin has tempted many researchers to study its biosynthetic route. A previous isotope-feeding experiment revealed that terretonin is derived from 1 and farnesyl diphosphate (FPP). Recently, we identified the biosynthetic gene cluster of terretonin (8) in the A. terreus NIH2624 genome and revealed, using a fungal heterologous expression system, that the PKS (trt4), PT (trt2) and FMO (trt8) genes, which are homologous to the pyripyropene A biosynthetic genes, are responsible for the production of epoxyfarnesyl-DMOA (3), a precursor of terretonin (Scheme 2). However, the CYC gene responsible for the cyclization of the farnesyl moiety of 3 remained unknown. Therefore, we set out to clarify the cyclization step of terretonin biosynthesis. Since there are many DMOA-derived meroterpenoids, significant knowledge about a number of compounds should be obtained by such a study. We first focused on trt1, which is homologous to the pyr4 gene and encoded upstream of trt2, as the CYC gene responsible for the cyclization of 3. To characterize the function of Trt1, we expressed trt1 along with trt4, trt2 and trt8 in the heterologous fungal host A. oryzae NSAR1, a quadruple auxotrophic mutant strain (niaD , sC , DargB, adeA ). By using this host and four expression vectors—pTAex3 harboring a argB marker, pPTRI harboring a ptrA marker, pUSA harboring a sC marker and pAdeA harboring a adeA marker —the five genes were coexpressed under the control of amyB promoter. The transformant was cultured in Czapek–Dox (CD) medium, supplemented with starch to induce expression. After three days, the culture supernatant and the mycelial extract were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). 1 and dihydroxyfarnesyl-DMOA (4), a hydrolyzed compound derived from 3, were observed, but in contrast to our expectaScheme 1. Representative fungal meroterpenoids.