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

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:PPi 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-hydroxy-phenyl-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, htyF, in the two separate ecd and hty clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product.

Structural diversity in echinocandin biosynthesis: the impact of oxidation steps and approaches toward an evolutionary explanation

“…It consisted of A-and B-type pneumocandins with various hydroxylation patterns at ornithine C-4 and C-5, as well as at homotyrosine C-4; however, there was no hydroxylation at homotyrosine C-3. These results nicely support the model for echinocandin biosynthesis proposed by the groups of Tang and Walsh [13,16]. They also show that the principle of echinocandin biosynthesis is well conserved, even in distantly related fungi.…”

“…In brief, the biosynthetic origin of the non-proteinogenic amino acids and the dimethylmyristate side chain was elucidated in the early 1990s by groups of Tang and Walsh disclosed the genes encoding echinocandin B biosynthesis in the genome of Aspergillus pachycristatus NRRL 11440 (formerly Emericella rugulosa) [13]. According to this study, the genes are arranged in two separate partial clusters (Ecd and Hty) located on different contigs of the assembly.…”

“…The first CAT module is selective for ornithine, which is initially N-acylated with the linoleic acid side chain. After assembly of the six amino acids, the peptide is cyclized by the terminal condensation domain (C T ) [13].…”

Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites

“…Recently, biosynthetic schemes have been proposed for echinocandin B (16,17) and for the pneumocandins (18) (Fig. 1).…”

At the Intersection of Chemistry, Biology, and Medicine

“…One was to decipher the timing and mechanism of the clinically useful echinocandin class of antifungal agents (51). A parallel study with Yi Tang et al (52) unearthed the logic that fungal NRPS assembly lines use to activate and insert the aromatic β amino acid anthranilate to build fused-ring systems ranging from tricyclic to heptacyclic scaffolds.…”