Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties.

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5-7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Universal Strategy to Develop Fluorogenic Probes for Lysine Deacylase/Demethylase Activity and Application in Discriminating Demethylation States.

Dynamically controlling the post-translational modification of the ε-amino groups of lysine residues is critical for regulating many cellular events. Increasing studies have revealed that many important diseases, including cancer and neurological disorders, are associated with the malfunction of lysine deacylases and demethylases. Developing fluorescent probes that are capable of detecting lysine deacylase and demethylase activity is highly useful for interrogating their roles in epigenetic regulation and diseases. Due to the distinct substrate recognition of these epigenetic eraser enzymes, designing a universal strategy for detecting their activity poses substantial difficulty. Moreover, designing activity-based probes for differentiating their demethylation states is even more challenging and still remains largely unexplored. Herein, we report a universal strategy to construct probes that can detect the enzymatic activity of epigenetic "erasers" through NBD-based long-distance intramolecular reactions. The probes can be easily prepared by installing the O-NBD group at the C-terminal residue of specific peptide substrates by click chemistry. Based on this strategy, detecting the activity of lysine deacetylase, desuccinylase, or demethylase with superior sensitivity and selectivity has been successfully achieved through single-step probe development. Furthermore, the demethylase probe based on this strategy is capable of distinguishing different demethylation states by both absorption and fluorescence lifetime readout. We envision that these newly developed probes will provide powerful tools to facilitate drug discovery in epigenetics in the future.

Biosynthetic Characterization, Heterologous Production, and Genomics-Guided Discovery of GABA-Containing Fungal Heptapeptides.

The biosynthetic gene cluster of γ-aminobutyric acid (GABA)-containing fungal cyclic heptapeptides unguisins A (1) and B (2) was identified in the fungus Aspergillus violaceofuscus CBS 115571. In vitro enzymatic reactions and gene deletion experiments revealed that the unguisin pathway involves the alanine racemase UngC to provide d-alanine, which is then accepted by the first adenylation domain of the nonribosomal peptide synthetase (NRPS) UngA. Intriguingly, the hydrolase UngD was found to transform unguisins into previously undescribed linear peptides. Subsequently, heterologous production of these peptides in Aspergillus oryzae was achieved, in which we established a methodology to readily introduce a large NRPS gene into the fungal host. Finally, genome mining revealed new unguisin congeners, each containing a (2R,3R)-ß-methylphenylalanine residue.

Depside Bond Formation by the Starter-Unit Acyltransferase Domain of a Fungal Polyketide Synthase.

Depsides are polyphenolic molecules comprising two or more phenolic acid derivatives linked by an ester bond, which is called a depside bond in these molecules. Despite more than a century of intensive research on depsides, the biosynthetic mechanism of depside bond formation remains unclear. In this study, we discovered a polyketide synthase, DrcA, from the fungus Aspergillus duricaulis CBS 481.65 and found that DrcA synthesizes CJ-20,557 (1), a heterodimeric depside composed of 3-methylorsellinic acid and 3,5-dimethylorsellinic acid. Moreover, we determined that depside bond formation is catalyzed by the starter-unit acyltransferase (SAT) domain of DrcA. Remarkably, this is a previously undescribed form of SAT domain chemistry. Further investigation revealed that 1 is transformed into duricamidepside (2), a depside-amino acid conjugate, by the single-module nonribosomal peptide synthetase DrcB.

BODI-Pt, a Green-Light-Activatable and Carboplatin-Based Platinum(IV) Anticancer Prodrug with Enhanced Activation and Cytotoxicity.

Platinum drugs are widely used in clinics to treat various types of cancer. However, a number of severe side effects induced by the nonspecific binding of platinum drugs to normal tissues limit their clinical use. The conversion of platinum(II) drugs into more inert platinum(IV) derivatives is a promising strategy to solve this problem. Some platinum(IV) prodrugs, such as carboplatin-based tetracarboxylatoplatinum(IV) prodrugs, are not easily reduced to active platinum(II) species, leading to low cytotoxicity in vitro. In this study, we report the design and synthesis of a carboplatin-based platinum(IV) prodrug functionalized with a boron dipyrromethene (bodipy) ligand at the axial position, and the ligand acts as a photoabsorber to photoactivate the platinum(IV) prodrug. This compound, designated as BODI-Pt, is highly stable in the dark but quickly activated under irradiation to release carboplatin and the axial ligands. A cytotoxic study reveals that BODI-Pt is effective under irradiation, with cytotoxicity 11 times higher than that in the dark and 39 times higher than that of carboplatin in MCF-7 cells. Moreover, BODI-Pt has been proven to kill cancer cells by binding to the genomic DNA, arresting the cell cycle at the G2/M phase, inducing oncosis, and generating ROS upon irradiation. In summary, we report a green-light-activatable and carboplatin-based Pt(IV) prodrug with improved cytotoxicity against cancer cells, and our strategy can be used as a promising way to effectively activate carboplatin-based platinum(IV) prodrugs.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation.

Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/α-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/α-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the α-ketoglutarate-binding ability.

Unique chemistry of non-heme iron enzymes in fungal biosynthetic pathways.

Covering: up to 2018 Non-heme iron enzymes are a versatile family of oxygenases that catalyze remarkable types of chemistry. This review highlights the intriguing chemistry of non-heme iron enzymes, especially those utilizing α-ketoglutarate (α-KG) as a co-substrate, in fungal secondary metabolism and aims to summarize how nature diversifies and complexifies natural products.