Biosynthesis of the Halogenated Mycotoxin Aspirochlorine in Koji Mold Involves a Cryptic Amino Acid Conversion

• Published in: Angewandte Chemie International Edition 2014-12, Vol.53 (49), p.13409-13413
• Main Authors: Chankhamjon, Pranatchareeya, Boettger-Schmidt, Daniela, Scherlach, Kirstin, Urbansky, Barbara, Lackner, Gerald, Kalb, Daniel, Dahse, Hans-Martin, Hoffmeister, Dirk, Hertweck, Christian
• Format: Article
• Language: English
• Subjects: Amino acids; Aspergillus oryzae; Aspergillus oryzae - chemistry; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Aspergillus oryzae - metabolism; Biosynthesis; Biosynthetic Pathways; CC cleavage; epidithiodiketopiperazine; Food Microbiology; Genetic Loci; halogenases; Halogenation; Koji; Marking; Molds; Mycotoxins; Mycotoxins - chemistry; Mycotoxins - genetics; Mycotoxins - metabolism; Peptide Synthases - chemistry; Peptide Synthases - genetics; Peptide Synthases - metabolism; Peptides; Spiro Compounds - chemistry; Spiro Compounds - metabolism; Toxins
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201407624

Aspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non‐ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin. A delicious riddle: The koji mold Aspergillus oryzae is used for the production of Asian delicacies like soy sauce and sake. The biosynthesis of the toxin aspirochlorine (1) in A. oryzae was elucidated by mutagenesis, in vitro adenylation, and isotope labeling. It was found that its peptide core is not assembled from Phe and Gly, but from two Phe units: one Phe undergoes chlorination and oxygenation whereas the other one is transformed into Gly.