Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway

Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects

Biosynthesis of sulfonamide and sulfamate antibiotics in actinomycete

Sulfonamides and sulfamates are a group of organosulfur compounds that contain the signature sulfamoyl structural motif. These compounds were initially only known as synthetic antibacterial drugs but were later also discovered as natural products. Eight highly potent examples have been isolated from actinomycetes to date, illustrating the large biosynthetic repertoire of this bacterial genus. For the biosynthesis of these compounds, several distinct and unique biosynthetic machineries have been discovered, capable to generate the unique S-N bond. For the creation of novel, second generation natural products by biosynthetic engineering efforts, a detailed understanding of the underlying enzyme machinery toward potent structural motifs is crucial. In this review, we aim to summarize the current state of knowledge on sulfonamide and sulfamate biosynthesis. A detailed discussion for the secondary sulfamate ascamycin, the tertiary sulfonamide sulfadixiamycin A, and the secondary sulfonamide SB-203208 is provided and their bioactivities and mode of actions are discussed.publishe

Spiro-Ring Formation is Catalyzed by a Multifunctional Dioxygenase in Austinol Biosynthesis

Austinol, a fungal meroterpenoid derived from 3,5-dimethylorsellinic acid, has a unique chemical structure with a remarkable spiro-lactone ring system. Despite the recent identification of its biosynthetic gene cluster and targeted gene-deletion experiments, the process for the conversion of protoaustinoid A (<b>2</b>), the first tetracyclic biosynthetic intermediate, to the spiro-lactone preaustinoid A3 (<b>7</b>) has remained enigmatic. Here we report the mechanistic details of the enzyme-catalyzed, stereospecific spiro-lactone ring-forming reaction, which is catalyzed by a non-heme iron-dependent dioxygenase, AusE, along with two flavin monooxygenases, the 5′-hydroxylase AusB and the Baeyer–Villiger monooxygenase AusC. Remarkably, AusE is a multifunctional dioxygenase that is responsible for the iterative oxidation steps, including the oxidative spiro-ring-forming reaction, to produce the austinol scaffold