**4. Fungal Non-Ribosomal Peptide Biosynthesis Involving Two Separate Pathway Crosstalk**

#### *4.1. The Biosynthesis of Spirotryprostatin A*

Many NPs bearing the spiro-carbon system exhibit potential biological activities. Intrigued by the privileged structure and usefulness of spiro carbon system, a great deal of attention has been paid to their catalytic enantioselective synthesis [39,40]. Understanding the mechanism of spiro-carbon biosynthesis and identifying the versatility of enzymes responsible for the spiro-carbon formation are also of great significance. Spirotryprostatins belong to non-ribosomal peptides that isolated from *A*. *fumigatus*, and known for their pharmaceutical importance and application in cancer treatment [41]. The formation of the spiro-ring moiety in spirotryprostatins remained unknown and aroused Watanabe and co-workers' interest to solve this mystery [42]. The authors utilized *S*. *cerevisiae* and *A*. *niger* as the heterologous hosts to efficiently express the whole biosynthetic pathways of spirotryprostatins, and obtained crucial intermediates to identify two pathways for spiro-carbon formation, namely an epoxide route catalyzed by the FMO FqzB and a radical route catalyzed by the cytochrome P450 FtmG (Figure 5) [42]. Spirotryprostatins possess the diketopiperazine frameworks, and show the structural similarity to fumitremorgins and fumiquinazolines, suggesting the peptide backbone of these compounds could be biosynthesized by the NRPS using L-proline and L-tryptophan as the biosynthetic precursors [43,44]. When *ftmA*-*E* five genes were expressed in *A*. *niger*, no intermediates featuring the spiro-carbon were isolated, implying other indispensable genes are needed to be introduced. Based on the prior researches about the biosynthetic mechanism of spiro-carbon formation [45–47], the authors creatively introduced the FMO FqzB-encoding gene *fqzB*, which is located within the fumiquinazoline BGC (designed as fqz BGC in this review), into the *A*. *niger*/ *ftmA*-*E* mutant, and successfully isolated pirotryprostatin A (**32**) (Figure 5). In vitro enzymatic assay revealed that FqzB could transform fumitremorgin C (**33**) into **32** by epoxidation mediated semipinacol-type rearrangement (Figure 5). These fascinating results not only emphasize the important function of FMOs as the intersection to trigger the coupling of two separate BGCs, but also highlight that the crosstalk between different biosynthetic pathways allows the structural diversification in NP biosynthesis.

**Figure 5.** The biosynthetic pathway of fungal non-ribosomal peptide spirotryprostatin A (**32**). The FMO FqzB from the *fqz* BGC catalyzes the formation of spiro-carbon in spirotryprostatin A.

#### *4.2. The Biosynthesis of Echinocandin B*

Echinocandins, a family of fungal lipohexapeptides, are firstly isolated from *Emericella rugulosa* NRRL 11440, and exhibit excellent antifungal activities to the opportunistic pathogenic *Candida* strains. Structural modifications of echinocandin B (**34**), especially for the fatty acid moiety, successfully led to the generation of FDA-approved drug anidulafungin, which is a semisynthetic derivative of **34** and contains a substituted terphenyl acyl chain. To better understand how microbes use simple precursors to synthesize complex NPs, Tang and co-workers performed the groundbreaking work to identify and characterize the BGC of echinocandin B [48]. Four nonproteinogenic amino acids including 4*R*,5*R*-dihydroxyl-L-ornithine, 3*S*-hydroxyl-4*S*-methyl-L-proline, 4*R*-hydroxyl-L-proline, and 3*S*,4*S*-dihydroxyl-L-homotyrosine, as well as a long chain fatty acyl amide were contained in echinocandin B (Figure 6). These unusual structural units implied an interesting biosynthetic mechanism of echinocandin B.

By bioinformatics analysis, the NRPS EcdA containing six modules was identified. Gene deletion of *ecdA* confirmed its vital role in the peptide backbone formation of echinocandin B. Other biosynthetic genes, such as *ecdI* encoding a fatty-acyl-AMP ligase (EcdI), *ecdG* and *ecdK* encoding two α-ketoglutarate dependent oxygenases, and *ecdH* encoding a heme-iron-dependent cytochrome P450 oxygenase, were all in proximity to *ecdA* to constitute the *ecd* BGC. However, the genes responsible for the biosynthesis of Lhomotyrosine were not present in the vicinity of *ecd* BGC, indicating a separate BGC should reside elsewhere in the genome. The putative 2-(4-hydroxybenzyl)-malic acid (**36**) is pro-

posed to be the biosynthetic intermediate of L-homotyrosine (**39**) (Figure 6). Considering the isopropyl-malate synthase (IPMS) is reported to catalyze the condensation of α-ketovalerate with acetyl-CoA in the leucine biosynthesis, one IPMS homology in *E*. *rugulosa* genome was proposed to catalyze the condensation of 4-hydroxyphenyl-pyruvate and acetate to form **36**. Using the IPMS gene from *Mycobacterium tuberculosis* as BLAST query, the authors successfully identified the *hty* BGC, which is about 42.5 kb away from the *ecd* BGC, encoding four enzymes for de novo generation of the special building block L-homotyrosine (Figure 6) [48]. The isopropyl malate dehydrogenase HtyA catalyzed aldol-type condensation of 4-hydroxyphenyl-pyruvate (**35**) and acetyl-CoA to form **36**. Then, the aconitase homology HtyD executed the isomerization of 36 to 3-(4-hydroxybenzyl)-malic acid (**37**). Thereafter, **37** underwent decarboxylation and oxidation to form 2-oxo-4-(4-hydroxybenzyl) butanoic acid (**38**) by isopropyl malate dehydrogenase homologue HtyC. Finally, the transaminase HtyB catalyzed the transamination of **38** to form **39**. In general, the biosynthesis of echinocandin B needs the coupling of two sperate BGCs, the *ecd* and *hty* BGCs. The *hty* BGC provides an important biosynthetic precursor L-homotyrosine which was recognized by the fourth A domain of the NRPS EcdA. Understanding the biosynthetic mechanism of echinocandin B will facilitate us to take advantage of synthetic biology techniques to bioengineer NRPSs to generate bioactive compounds [49–51].

**Figure 6.** The biosynthetic pathway of fungal non-ribosomal peptide echinocandin B (**34**). The separate *hty* BGC is responsible for the L-homotyrosine moiety formation.
