Structure and localization of cyclosporin synthetase, the key enzyme of cyclosporin biosynthesis in Tolypocladium inflatum

Hoppert, Michael; Gentzsch, Carsten; Schörgendorfer, Kurt

doi:10.1007/s002030100324

Cited by 66 publications

(48 citation statements)

References 29 publications

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“…There is evidence to suggest that many secondary metabolite biosynthetic pathways in fungi are restricted to specific organelles, as shown for cyclosporine, penicillin, and aflatoxin biosynthesis (51)(52)(53)(54)(55)(56). Recently specific synthesis vesicles, aflatoxisomes (57), were discovered in Aspergillus parasiticus.…”

Section: Discussionmentioning

confidence: 99%

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Frandsen

Schütt

Lund

et al. 2011

Journal of Biological Chemistry

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Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated ␥-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to-aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.The plant pathogenic fungus Fusarium graminearum (teleomorph Gibberella zeae) is capable of producing a plethora of secondary metabolites, many of which belong to the polyketide class of compounds, such as the mycotoxins zearalenone, fusarin C, and aurofusarin (1). The F. graminearum genome encodes 15 polyketide synthases (PKS) 2 (2, 3), of which FgPKS12 is the progenitor of nor-rubrofusarin/rubrofusarin/aurofusarin (4, 5), FgPKS4 and FgPKS13 are the progenitors of zearalenone (1, 6), FgPKS10 is the progenitor of fusarin C (7, 8), and FgPKS3 is the progenitor of an uncharacterized black/purple perithecial pigment (8). PKS genes are typically found in gene clusters that comprise genes encoding transcription factor, transporters, and tailoring enzymes required for biosynthesis of the final product (1, 4 -6). In addition to genes encoding well characterized classes of enzymes, the clusters typically also include orphan genes that encode proteins annotated as "hypothetical" or "conserved hypothetical proteins," signifying that no similarity to any previously characterized protein has been found. In the case of F. graminearum, more than half of all gene models found in the Fusarium graminearum Genome Database from the Munich Information Center for Protein Sequences (MIPS) fall into either of these two categories (9).Functional characterization of hypothetical proteins is challenging, but those belonging to secondary metabolite gene clusters provide a unique opportunity because their location suggests a function in the respective biosynthetic pathways. The PKS12 gene cluster in F. graminearum, which is responsible for biosyn...

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Section: Discussionmentioning

confidence: 99%

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Frandsen

Schütt

Lund

et al. 2011

Journal of Biological Chemistry

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“…Comparison with an authentic sample of rhizonin A and the MS n fragmentation pattern proved the presence of the toxin. The structure of rhizonin suggests that its biosynthesis affords an NRPS, similar to enzymes that are involved in the formation of structurally related fungal cyclopeptides, e.g., the immunosuppressive cyclosporine (8,21,22). Typically, NRPSs represent giant multidomain enzymes with a modular architecture.…”

Section: Microsporus Var Oligosporus Isolates) (9)mentioning

confidence: 99%

Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts

Partida‐Martínez

Looß

Ishida

et al. 2007

Appl Environ Microbiol

143

115

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Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this "first mycotoxin from lower fungi" is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.

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“…Little is known about the structural elements that facilitate this specific interaction. In particular, this interaction might be less efficient in the heterologous host E. coli because of different cellular conditions, unknown factors, or improper localization to the cell membrane, which has been found for many NRPSs in their natural producer strains (19). We therefore used a construct in which the structural genes of TycA and TycB1 are fused to one open reading frame.…”

Section: Strategymentioning

confidence: 99%

In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

Gruenewald

Mootz

Stehmeier

et al. 2004

Appl Environ Microbiol

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Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

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Structure and localization of cyclosporin synthetase, the key enzyme of cyclosporin biosynthesis in Tolypocladium inflatum

Cited by 66 publications

References 29 publications

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts

In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

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