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.

C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.

Reprogramming of the NRPS/PKS assembly line is an attractive method for the production of new bioactive molecules. However, it is usually hampered by the loss of intimate domain/module interactions required for the precise control of chain transfer and elongation reactions. In this study, we first establish heterologous expression systems of the unique antimycin-type cyclic depsipeptides: JBIR-06 (tri-lactone) and neoantimycin (tetra-lactone), and engineer their biosyntheses by taking advantage of bioinformatic analyses and evolutionary insights. As a result, we successfully accomplish three manipulations: (i) ring contraction of neoantimycin (from tetra-lactone to tri-lactone), (ii) ring expansion of JBIR-06 (from tri-lactone to tetra-lactone), and (iii) alkyl chain diversification of JBIR-06 by the incorporation of various alkylmalonyl-CoA extender units, to generate a set of unnatural derivatives in practical yields. This study presents a useful strategy for engineering NRPS-PKS module enzymes, based on nature’s diversification of the domain and module organizations.

Sulfonamide is present in many important drugs, due to its unique chemical and biological properties. In contrast, naturally occurring sulfonamides are rare, and their biosynthetic knowledge are scarce. Here we identify the biosynthetic gene cluster of sulfonamide antibiotics, altemicidin, SB-203207, and SB-203208, from Streptomyces sp. NCIMB40513. The heterologous gene expression and biochemical analyses reveal unique aminoacyl transfer reactions, including the tRNA synthetase-like enzyme SbzA-catalyzed L-isoleucine transfer and the GNAT enzyme SbzC-catalyzed β-methylphenylalanine transfer. Furthermore, we elucidate the biogenesis of 2-sulfamoylacetic acid from L-cysteine, by the collaboration of the cupin dioxygenase SbzM and the aldehyde dehydrogenase SbzJ. Remarkably, SbzM catalyzes the two-step oxidation and decarboxylation of L-cysteine, and the subsequent intramolecular amino group rearrangement leads to N-S bond formation. This detailed analysis of the aminoacyl sulfonamide antibiotics biosynthetic machineries paves the way toward investigations of sulfonamide biosynthesis and its engineering.