Ustiloxins represented by ustiloxin B (1) were toxic fungal ribosomal peptides, isolated from rice false smut caused by the pathogenic fungus Ustilaginoidea virens (Figure 1).1-3These family members, including a structurally related phomopsin and asperipin-2a (Figure 1), exhibit potent antimitotic activity and inhibit microtubule assembly. Of particular structural interest is a maclocyclic structure constructed by an oxidative cyclization between the amino acid side chains. Recently, a biosynthetic gene cluster (ust) of 1was identified in the genome of Aspergillus flavus using a sequence motif-independent de novo detection algorithm (MIDDAS-M).4,5 The cluster contained a unique precursor protein, UstA, that has 16 repeated Tyl-Ala-Ile-Gly (YAIG) sequences. Recently, we elucidated the biosynthetic machinery of 1 through heterologous expression and in vitro studies.6 The results confirmed the oxidation enzymes harboring a DUF3328 motif for the macrocylization and unique modification enzymes for the amino acid-like side chain on the aromatic ring. In this presentation, we will discuss the biosynthesis of the first ribosomal peptide produced by filamentous fungi.
Proposed biosynthetic pathway of ustiloxins
Previous gene knockout experiments showed eleven genes were responsible for the ustiloxin biosynthesis.7 LC-MS analysis of the extracts from five mutants, DustM, DustC, DustF1, DustF2, and DustD, showed the accumulation of ustiloxin derivatives (2-6). Except known ustiloxins, ustiloxin F (3) and ustiloxin C (6), our collaborators determined the structure of novel ustiloxins, N-desmethylustiloxin F (2), S-deoxyustiloxin H (4), and ustiloxin H (5).6These data enabled us to speculate the functions of the ustMCF1F2D genes. For the last transformation with UstD, condensation of a C3 nucleophile with an aldehyde form 8 of 6is more likely to be involved. Taken together, we proposed a biosynthetic pathway for 1 as shown in Scheme 1.6
Cyclic peptide formation catalyzed by unprecedented oxidation enzymes
The gene knockout experiments of ustQYaYbindicated that these enzyme genes were involved in the characteristic cyclic peptide formation because the corresponding mutants completely abolished production of 2. The ustQ gene displays the highest similarity to a tyrosinase which usually catalyses an oxidation of tyrosine and dopamine. The ustYa and ustYb genes exhibit no homology with functionally characterized enzymes but have a common DUF3328 motif. For functional characterization of these genes, we conducted heterologous expression in Aspergillus oryzae.8We transformed the wild-type NSAR1 strain by applying the “tandem transformation8” strategy with different combination of plasmids, and three transformants, AO-ustAQYa, AO-ustAQYb, and AO-ustAQYaYb, were obtained (Figure 2). Although AO-ustAQYaand AO-ustAQYb produced no cyclic- and linear peptides, AO-ustAQYaYbgave 2 (Figure 2). Additional introduction of ustYb into AO-ustAQYa resulted in the production of 2 (Figure 2), suggesting that both UstY enzymes are responsible for the oxidative cyclization. The successful in vivo reconstitution of the biosynthetic machinery of 2further confirmed the importance of the three oxidation enzymes. Given the results of the gene inactivation studies, we speculated that the UstA protein is digested into 16 pieces of trideca-/tetradeca-peptides by intrinsic Kex2 prot
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