Identification and Characterization of the Echinocandin B Biosynthetic Gene Cluster from Emericella rugulosa NRRL 11440

Journal of the American Chemical Society (Impact Factor: 11.44). 10/2012; 134(40):16781-90. DOI: 10.1021/ja307220z

ABSTRACT Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4R,5R-dihydroxy-L-ornithine, 4R-hydroxyl-L-proline, 3S,4S-dihydroxy-L-homotyrosine, and 3S-hydroxyl-4S-methyl-L-proline. We report here the biosynthetic gene cluster of echinocandin B 1 from Emericella rugulosa NRRL 11440 containing genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. We showed EcdI activates linoleate as linoleyl-AMP and installs it on the first thiolation domain of EcdA. We have also established through ATP-PP(i) exchange assay that EcdA loads L-ornithine in the first module. A separate hty gene cluster encodes four enzymes for de novo generation of L-homotyrosine from acetyl-CoA and 4-hydroxyphenyl-pyruvate is found from the sequenced genome. Deletions in the ecdA, and htyA genes validate their essential roles in echinocandin B production. Five predicted iron-centered oxygenase genes, ecdG, ecdH, ecdK, htyE, and htyF, in the two separate ecd and hty clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product.

    • "Interestingly, the best results were obtained with L-Tyr and L-Phe (15 g/l) (Tóth 2012), which are good sources of 4- hydroxyl-phenyl-pyruvate, the precursor of homoTyr synthesis (Cacho et al. 2012). Importantly, plant oils also enhance echinocandin B production (Boeck and Kastner 1981; Tóth et al. 2011; Tóth 2012) and are generally used in echinocandin fermentations to enhance the formation of the acyl side-chains of the antimycotics. "
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    ABSTRACT: The first echinocandin-type antimycotic (echinocandin B) was discovered in the 1970s. It was followed by the isolation of more than 20 natural echinocandins. These cyclic lipo-hexapeptides are biosynthesized on non-ribosomal peptide synthase complexes by different ascomycota fungi. They have a unique mechanism of action; as non-competitive inhibitors of β-1,3-glucan synthase complex they target the fungal cell wall. Results of the structure-activity relationship experiments let us develop semisynthetic derivatives with improved properties. Three cyclic lipohiexapeptides (caspofungin, micafungin and anidulafungin) are currently approved for use in clinics. As they show good fungicidal (Candida spp.) or fungistatic (Aspergillus spp.) activity against the most important human pathogenic fungi including azole-resistant strains, they are an important addition to the antifungal armamentarium. Some evidence of acquired resistance against echinocandins has been detected among Candida glabrata strains in recent years, which enhanced the importance of data collected on the mechanism of acquired resistance developing against the echinocandins. In this review, we show the structural diversity of natural echinocandins, and we summarize the emerging data on their mode of action, biosynthesis and industrial production. Their clinical significance as well as the mechanism of natural and acquired resistance is also discussed.
    Applied Microbiology and Biotechnology 03/2013; 97(8). DOI:10.1007/s00253-013-4761-9 · 3.81 Impact Factor
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    ABSTRACT: L-Homophenylalanine (L-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemo-enzymatic route of synthesis is fully developed, we investigated microbial production of L-Hph to explore the possibility of a more efficient and sustainable approach to L-Hph production. We hypothesized that L-Hph is synthesized from L-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatic analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD-expressing plasmids and achieved the fermentative production of L-Hph from L-Phe. To our knowledge, this is a first identification of the genes responsible for the homophenylalanine synthesis in any organisms. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA, hphB, and hphCD, which increased the yield to ∼630 mg/L. The L-Hph biosynthesis and L-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show a tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the L-Hph-producing strain was examined, which showed m-Fluorophenylalanine, o-Fluorophenylalanine, and L-Tyrosine were accepted as substrates, and that the corresponding homoamino acids were generated.
    Applied and Environmental Microbiology 01/2013; DOI:10.1128/AEM.03596-12 · 3.95 Impact Factor
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    ABSTRACT: The echinocandins are a small group of fungal N-acylated cyclic hexapeptides that are fungicidal for candida strains and fungistatic for aspergilli by targeting cell wall 1,3-β-glucan synthases. The side chains of all six amino acid building blocks have hydroxyl groups, including the nonproteinogenic 4R,5R-dihydroxy-Orn1, 4R-OH-Pro3, 3S, 4S-dihydroxy homoTyr4 and 3S-OH-4S-Me-Pro6. The echinocandin (ecd) gene cluster contains two predicted nonheme mononuclear iron oxygenase genes (ecdG,K) and one encoding a P450 type heme protein (ecdH). Deletion of the ecdH gene in the producing Emericella rugulosa generates an echinocandin scaffold (echinocandin D) lacking both hydroxyl groups on Orn1. Correspondingly, the ΔecdG strain failed to hydroxylate C3 of the homoTyr residue, and purified EcdG hydroxylated free L-homoTyr at C3. The ΔecdK strain failed to generate mature echinocandin unless supplemented with either 4-Me-Pro or 3-OH-4-Me-Pro, indicating blockage of a step upstream of MePro formation. Purified EcdK is a Leu 5-hydroxylase, acting iteratively at C5 to yield γ-Me-Glu-γ-semialdehydein equilibrium with the cyclic imine product. Evaluation of deshydroxyechinocandin scaffolds in in vitro anticandidal assays revealed up to 5 fold loss of potency for the ΔecdG scaffolds, but a 3-fold gain of potency for the ΔecdH scaffold, in line with prior results on deoxyechinocandin homologs.
    Journal of the American Chemical Society 02/2013; 135(11). DOI:10.1021/ja312572v · 11.44 Impact Factor
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