Identifying the minimal enzymes required for anhydrotetracycline biosynthesis.
The cyclohexenone ring A of tetracyclines exhibits unique structural features not observed among other aromatic polyketides. These substitutions include the C2 primary amide, C4 dimethylamine, and the C12a tertiary alcohol. Here we report the identification and reconstitution of the minimum set of enzymes required for the biosynthesis of anhydrotetracycline (ATC, 5), the first intermediate in the tetracycline biosynthetic pathway that contains the fully functionalized ring A. Using a combination of in vivo and in vitro approaches, we confirmed OxyL, OxyQ, and OxyT to be the only enzymes required to convert 6-methylpretetramid 1 into 5. OxyL is a NADPH-dependent dioxygenase that introduces two oxygen atoms into 1 to yield the unstable intermediate 4-keto-ATC 2. The aminotransferase OxyQ catalyzes the reductive amination of C4-keto of 2, yielding 4-amino-ATC 3. Furthermore, the N, N-dimethyltransferase OxyT catalyzes the formation of 5 from 3 in a (S)-adenosylmethionine (SAM)-dependent manner. Finally, a "non-natural" anhydrotetracycline derivative was generated, demonstrating that our heterologous host/vector pair can be a useful platform toward the engineered biosynthesis of tetracycline analogues.
Zhang
Wenjun
W
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, USA.
Watanabe
Kenji
K
Cai
Xiaolu
X
Jung
Michael E
ME
Tang
Yi
Y
Zhan
Jixun
J
D049308:Dioxygenases / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000378:metabolism; D013754:Tetracyclines / Q000096:biosynthesis*; D000637:Transaminases / Q000378:metabolism
D013754:Tetracyclines; C016229:4-epianhydrotetracycline; D049308:Dioxygenases; D008780:Methyltransferases; D000637:Transaminases