Completing our understanding of tetracycline biosynthesis: the enzymatic basis of the F420‐dependent final reduction step.

Abstract

Tetracyclines are important broad‐spectrum antibiotics. While the discovery of tetracyclines date back to the 1940s, our understanding of the biosynthesis of tetracyclines has only been investigated in recent years. One unknown step in the pathway is the conversion of 5a, 11a‐dehydrotetracycline to either oxytetracycline in Streptomyces rimosus or tetracycline in Streptomyces aureofaciens. The reduction of the 5a–11a double bond is likely catalyzed by a F420‐dependent enzymes, however, the enzymatic basis for this reaction remains unresolved. Using a heterologous expression platform as a starting point, we discovered a small, previously unknown enzyme in both the oxy and ctc pathways that is responsible for this reduction. In vitro reconstitution using this small enzymes, in conjunction with the anhydrotetracycline oxygenase, showed that anhydrotetracycline can be converted to the final products. We also provide biochemical and structural explanation for the differential formation of oxytetracycline and tetracycline from the respective pathways. The result of this work therefore completes our understanding of the tetracycline pathways and pave the way for engineered biosynthesis of analogs.