Formation of Bridged Disulfide in Epidithiodioxopiperazines.

Epidithiodioxopiperazine (ETP) alkaloids, featuring a 2,5-diketopiperazine core and transannular disulfide bridge, exhibit a broad spectrum of biological activities. However, the structural complexity has prevented efficient chemical synthesis and further clinical research. In the past few decades, many achievements have been made in the biosynthesis of ETPs. Here, we discuss the biosynthetic progress and summarize them as two comprehensible metabolic principles for better understanding the complex pathways of α, α'- and α, ß'-disulfide bridged ETPs. Specifically, we systematically outline the catalytic machineries to install α, α'- and α, ß'-disulfide by flavin-containing oxygenases. This concept would contribute to the medical and industrial applications of ETPs.

An ortho-Quinone Methide Mediates Disulfide Migration in the Biosynthesis of Epidithiodiketopiperazines.

The transannular disulfide functions as a key structural element imparting diverse biological activities to epidithiodiketopiperazines (ETPs). Although mechanisms were proposed in previous studies, α,ß'-disulfide formation in ETPs is not well-determined owing to the failure to identify the hypothetical intermediate. Herein, we characterize the key ortho-quinone methide (o-QM) intermediate and prove its involvement in the carbon-sulfur migration from an α,α'- to an α,ß'-disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD-dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of the α,ß'-disulfide was initiated by Gln140 triggering proton abstraction for generation of the essential o-QM intermediate, accompanied by ß'-acetoxy elimination. Subsequent attack on the α,α'-disulfide by Cys137 led to disulfide migration and spirofuran formation. This study expands the biocatalytic toolbox for transannular disulfide formation and sets the stage for the targeted discovery of bioactive ETPs.

Pretrichodermamide A Biosynthesis Reveals the Hidden Diversity of Epidithiodiketopiperazines.

Fungal epidithiodiketopiperazines (ETPs) possess large structural diversity and complexity due to modifications of the cyclodipeptide skeleton. Elucidation of the biosynthetic pathway of pretrichodermamide A (1) in Trichoderma hypoxylon revealed a flexible catalytic machinery of multiple enzymes for generating ETP diversity. Seven tailoring enzymes encoded by the tda cluster are involved in 1 biosynthesis, that is, four P450s TdaB and TdaQ for 1,2-oxazine formation, TdaI for C7'-hydroxylation, and TdaG for C4, C5-epoxidation, two methyltransferases TdaH for C6'- and TdaO for C7'-O-methylation, and a reductase TdaD for furan opening. Gene deletions led to the identification of 25 novel ETPs, including 20 shunt products, indicating the catalytic promiscuity of Tda enzymes. Particularly, TdaG and TdaD accept various substrates and catalyze regiospecific reactions at different stages of 1 biosynthesis. Our study not only uncovers a hidden library of ETP alkaloids, but also helps to understand the hidden chemical diversity of natural products by pathway manipulation.

Quantitative characterization of filamentous fungal promoters on a single-cell resolution to discover cryptic natural products.

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods. Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution. Using this quantitative method, we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format. The strengths of identified promoters covered a 37-fold range by flow cytometry. PzipA and PsltA were identified as the strongest promoters, which were 2.9- and 1.5-fold higher than that of the commonly used constitutive promoter PgpdA. Thus, we applied PzipA and PsltA to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A. nidulans. The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives, namely, fumiganins A and B. Our method provides an innovative strategy for natural product discovery in fungi.

UvSorA and UvSorB Involved in Sorbicillinoid Biosynthesis Contribute to Fungal Development, Stress Response and Phytotoxicity in Ustilaginoidea virens.

Ustilaginoidea virens (teleomorph: Villosiclava virens) is an important fungal pathogen that causes a devastating rice disease. It can produce mycotoxins including sorbicillinoids. The biosynthesis and biological functions of sorbicillinoids have not been reported in U. virens. In this study, we identified a sorbicillinoid biosynthetic gene cluster in which two polyketide synthase genes UvSorA and UvSorB were responsible for sorbicillinoid biosynthesis in U. virens. In ∆UvSorA and ∆UvSorB mutants, the mycelial growth, sporulation and hyphal hydrophobicity were increased dramatically, while the resistances to osmotic pressure, metal cations, and fungicides were reduced. Both phytotoxic activity of rice germinated seeds and cell wall integrity were also reduced. Furthermore, mycelia and cell walls of ∆UvSorA and ∆UvSorB mutants showed alterations of microscopic and submicroscopic structures. In addition, feeding experiment showed that sorbicillinoids could restore mycelial growth, sporulation, and cell wall integrity in ∆UvSorA and ∆UvSorB mutants. The results demonstrated that both UvSorA and UvSorB were responsible for sorbicillinoid biosynthesis in U. virens, and contributed to development (mycelial growth, sporulation, and cell wall integrity), stress responses, and phytotoxicity through sorbicillinoid mediation. It provides an insight into further investigation of biological functions and biosynthesis of sorbicillinoids.

Characterization of a NRPS-like Protein from Pestalotiopsis fici for Aldehyde Generation.

Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from Pestalotiopsis fici and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from Saccharomyces cerevisiae. In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (1-28) revealed the corresponding aldehyde formation in 14 cases (1-14). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, 4) and vanillic acid (10). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.

Rapid and Accurate Screening of Lysine-Producing Edible Mushrooms via the Homocitrate Synthase Gene as a Universal Molecular Marker.

Edible mushrooms are important nutraceutical sources of foods and drugs, which can produce various nutritional ingredients including all essential amino acids. The method of rapid screening for the strains producing specific functional components is very indispensable. Homocitrate synthase is one of the key enzymes in the α-aminoadipate pathway for lysine biosynthesis and has preferable sequence conservation in Agaricales. Based on the blast of homocitrate synthase homologous genes of strains of Agaricales, we achieved combinations of degenerate primers as molecular markers to rapidly screen the lysine-producing edible mushrooms. The experimental results revealed that the consistency between PCR amplification and HPLC analysis attained 82 and 75% in strains of Agaricales and Polyporales, respectively. The finding showed that the molecular marker has higher universality for screening edible mushroom resources of Agaricales. This PCR-based approach shows excellent potential in evaluating and discriminating edible wild-grown mushrooms with high lysine content in Agaricales.

A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids.

Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.