Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self-protection is necessary to avoid self-harm resulting from over-accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over-produced, the non-enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for selfprotection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.Microbial natural products (NPs) have diverse bioactivities. They protect producers from predators, antagonize the growth of other species or hosts, and help to compete for the ecological niche. Some bioactive NPs, such as antibiotics and mycotoxins, are important to compete against other organisms within the same ecological space. Therefore, they are usually produced abundantly to maximize the competitive capacity of their producers. [1] It has been suggested that the efficient release of NP backbones from the enzymatic assembly lines has contributed significantly to the NP yields. [2] The main types of NPs in nature, such as polyketides (PKs), non-ribosomal peptides (NRPs), ribosomally synthesized and post-translationally modified peptides (RiPPs), and terpenes, are released through cyclization, hydrolysis and reduction. [3] These reactions can be catalyzed by assembly enzyme-fused domains or stand-alone enzymes, such as condensation domains, thioesterases, reductases and product templates. [4] In bacteria, PKs are also liberated by the stand-alone small cyclases, [5] which belong to the nuclear transport factor 2 (NTF2)-like superfamily SnoaL-2 proteins. These proteins have also been shown to act as postline tailoring enzymes, catalyzing highly selective [6+4] cycloaddition and polyether formation. [6] In fungi, an NTF2like superfamily protein has been reported to catalyze semipinacol rearrangement. [7] But their ability to act as cyclases is poorly characterized. Meanwhile, NP producers, have evolved exquisite strategies to avoid self-harm from bioactive NPs, [8] in particular if producers also contain the conserved target of NPs. These detoxification strategies include export by transporters or subcellular trafficking, neutralization by duplication of self-resistance target proteins, and NP inactivation by enzyme-catalyzed chemical modifications. [1b, 8, 9] However, the regulatory coordination between the backbone release and detoxification in fungi for the physiologically tolerable pr...

Mycotoxins have substantial impacts on agricultural production and food preservation. Some have high similarities in bioactivity but subtle differences on structures from various fungal producers. Understanding of their complex cross-biosynthesis will provide new insights into enzyme functions and food safety. Here, based on structurally related mycotoxins, such as aurovertins, asteltoxin, and citreoviridin, we showed that methyltransferase (MT)-catalyzed methylation is required for efficient oxidation and polyketide stability. MTs have broad interactions with polyketide synthases and flavin-containing monooxygenases (FMOs), while MT AstB is required for FMO AstC functionality in vivo. FMOs have common catalysis on pyrone–polyene intermediates but different catalytic specificity and efficiency on oxidative intermediates for the selective production of more toxic and complex mycotoxins. Thus, the subtle protein interaction and elaborate versatile catalysis of biosynthetic enzymes contribute to the efficient and selective biosynthesis of these structure-related mycotoxins and provide the basis to re-evaluate and control mycotoxins for agricultural and food safety.

Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self-protection is necessary to avoid self-harm resulting from over-accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over-produced, the non-enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for selfprotection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.Microbial natural products (NPs) have diverse bioactivities. They protect producers from predators, antagonize the growth of other species or hosts, and help to compete for the ecological niche. Some bioactive NPs, such as antibiotics and mycotoxins, are important to compete against other organisms within the same ecological space. Therefore, they are usually produced abundantly to maximize the competitive capacity of their producers. [1] It has been suggested that the efficient release of NP backbones from the enzymatic assembly lines has contributed significantly to the NP yields. [2] The main types of NPs in nature, such as polyketides (PKs), non-ribosomal peptides (NRPs), ribosomally synthesized and post-translationally modified peptides (RiPPs), and terpenes, are released through cyclization, hydrolysis and reduction. [3] These reactions can be catalyzed by assembly enzyme-fused domains or stand-alone enzymes, such as condensation domains, thioesterases, reductases and product templates. [4] In bacteria, PKs are also liberated by the stand-alone small cyclases, [5] which belong to the nuclear transport factor 2 (NTF2)-like superfamily SnoaL-2 proteins. These proteins have also been shown to act as postline tailoring enzymes, catalyzing highly selective [6+4] cycloaddition and polyether formation. [6] In fungi, an NTF2like superfamily protein has been reported to catalyze semipinacol rearrangement. [7] But their ability to act as cyclases is poorly characterized. Meanwhile, NP producers, have evolved exquisite strategies to avoid self-harm from bioactive NPs, [8] in particular if producers also contain the conserved target of NPs. These detoxification strategies include export by transporters or subcellular trafficking, neutralization by duplication of self-resistance target proteins, and NP inactivation by enzyme-catalyzed chemical modifications. [1b, 8, 9] However, the regulatory coordination between the backbone release and detoxification in fungi for the physiologically tolerable pr...