The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5'-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi

The biosynthetic machinery of the first fungal ribosomally synthesized and post‐translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side‐chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.

Mycotoxin cyclochlorotine (1) and structurally related astins are cyclic pentapeptides containing unique nonproteinogenic amino acids, such as β-phenylalanine, l-allo-threonine, and 3,4-dichloroproline. Herein, we report the biosynthetic pathway for 1, which involves intriguing tailoring processes mediated by DUF3328 proteins, including stereo- and regiospecific chlorination and hydroxylation and intramolecular O,N-transacylation. Our findings demonstrate that DUF3328 proteins, which are known to be involved in oxidative cyclization of fungal ribosomal peptides, have much higher functional diversity than previously expected.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.Phomopsin A (1, Figure 1) [1] is a mycotoxin produced by the fungal pathogen Phomopsis leptostromiformis (Diaporthe toxica), which infects lupins and causes lupinosis disease in livestock. [2] It is also a potent antimitotic agent that binds to tubulin with high affinity. [3,4] Regarding its chemical structure and biosynthesis, 1 is a hexapeptide classified as a ribosomally synthesized and posttranslationally modified peptide (RiPP) [5,6] consisting of a 13-membered macrocyclic ether, chlorinated tyrosine (Tyr), and dehydroamino acids. [1] In 2016, Ding et al. reported a biosynthetic gene cluster of 1 and

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.Phomopsin A (1, Figure 1) [1] is a mycotoxin produced by the fungal pathogen Phomopsis leptostromiformis (Diaporthe toxica), which infects lupins and causes lupinosis disease in livestock. [2] It is also a potent antimitotic agent that binds to tubulin with high affinity. [3,4] Regarding its chemical structure and biosynthesis, 1 is a hexapeptide classified as a ribosomally synthesized and posttranslationally modified peptide (RiPP) [5,6] consisting of a 13-membered macrocyclic ether, chlorinated tyrosine (Tyr), and dehydroamino acids. [1] In 2016, Ding et al. reported a biosynthetic gene cluster of 1 and