**3. Fungal Meroterpenoids Biosynthesis Involving Two Separate Pathway Crosstalk** *The Biosynthesis of Austinol*

Meroterpenoids are an important class of fungal NPs [28], some of them have been developed as the drug candidate for treatment of Alzheimer's disease such as territrem [29], and the drug lead for insecticide such as pyripyropene A [30]. Fungal meroterpenoids are usually biosynthesized by a single BGC which encodes a polyketide synthase, a terpene cyclase, a prenyltransferase, and other essential tailoring enzymes to produce polyketide and terpenoid precursors [31]. Deletion of *sumO* gene, encoding the small ubiquitin-like protein SUMO, significantly altered the profiles of secondary metabolites in *A*. *nidulans*. Austinol (**23**) and dehydroaustinol (24) were identified from the ∆*sumO* mutant [32]. Nielsen et al. first reported the NR-PKS AusA responsible for the formation of polyketide precursor 3,5-dimethylorsellinic acid (DMOA) (**25**) in austinol biosynthesis [33]. This conclusion was also verified by Wang et al. [34]. Interestingly, Wang and co-workers found that no prenyltransferase gene was located near NR-PKS gene *ausA*, which suggested that the genes responsible for the biosynthesis of **23** and **24** might be separated in the genome of *A*. *nidulans* LO2026 [34]. Using the UbiA sequence as a query to blast the prenyltransferase homologs in the genome of *A*. *nidulans*, Wang et al. found the top two candidate genes *AN9259.4* (designated as *ausN*, on chromosome VIII) and *AN8142.4*. Deletion of *ausN* abolished the production of **23** and **24**, and accumulated the polyketide precursor **25**. By a set of gene deletions around *ausA* and *ausN*, they identified the two separate BGCs: the BGC A containing the necessary genes *ausA*-*D* and the BGC B consisting of the biosynthetic genes *ausE*-*N*. The biosynthetic pathway for **23** and **24** was also proposed (Figure 4). Firstly, the polyketide synthase AusA is responsible for the formation of **25**, then the aromatic prenyltransferase AusN catalyzed the C-alkylation of **25** using farnesyl pyrophosphate to form intermediate **26**. The epoxidase AusM catalyzed the epoxidation of the prenylated polyketide intermediate **26**, followed by cyclization catalyzed by a terpene cyclase AusL to form the tetracyclic intermediate **27**. The formation mechanism of the lactone system and spiro-ring in compound **28** has been investigated by Abe group [35]. The co-operation of the non-heme iron-dependent dioxygenase AusE, the hydroxylase AusB, and the Baeyer–Villiger monooxygenase AusC transformed the substrate **27** into **28** [35]; The hypothetical protein AusJ might be responsible for the acid-catalyzed ketorearrangement and ring contraction of the tetraketide portion in intermediate **28** to generate intermediate **29**. The authors speculated that the AusK is responsible for reducing the C-50 keto of **29** to hydroxyl group, and the hypothetical protein AusH might function as an accessory enzyme collaboratively working with AusK to alter AusK stereospecificity for its product **30**. The Baeyer−Villiger monooxygenase AusI inserted an oxygen atom between the C-40 and vicinal carbon at C-30 of **30** to create a lactone ring in **31**. Finally, the P450 monooxygenase AusG might catalyze the C-11 hydroxylation of **31** to form final product austinol (**23**) (Figure 4).

DMOA-derived fungal meroterpenoids possess complicated structures and attracted researchers' attention to investigate the biosynthetic pathway. Some fungal meroterpenoids such as anditomin and andrastin have been studied in detail [36–38]. All of the necessary genes for anditomin or andrastin biosynthesis are clustered in one single BGC. However, the biosynthesis of austinol and dehydroaustinol in *A*. *nidulans* LO2026 needs the pathway

crosstalk between BGC A and BGC B. This provides an intriguing and valuable insight that fungi could use a variety of strategies to expand the skeletal diversification and subtlety regulate the crosstalk between separate biosynthetic pathways to multiply the number of NPs produced by these BGCs.

**Figure 4.** The biosynthetic pathway of fungal meroterpenoids austinol (**23**) and dehydroaustinol (**24**) in *A*. *nidulans* LO2026.
