The role of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus

Enej Kuer, Nigel Coates, Iain Challis, Matt Gregory, Barrie Wilkinson, Rose Sheridan, and Hrvoje Petkovi^*

Biotica Technology Limited, Chesterford Research Park, CB10 1XL Essex, UK

^* To whom correspondence should be addressed. Email: hrvoje.petkovic{at}bf.uni-lj.si.

	Abstract

Rapamycin is an important macrocyclic polyketide produced by Streptomyces hygroscopicus showing immunosuppressive, antifungal and antitumor activities as well as displaying anti-inflammatory and neuroregenerative properties. The immense pharmacological potential of rapamycin has led to the production of an array of analogues, including through genetic engineering of the rapamycin biosynthetic gene cluster. This cluster contains several putative regulatory genes. Based on DNA sequence analysis, genes rapH and rapG revealed high similarities with two different families of transcriptional activators: LAL and AraC, respectively. Over-expression of either gene resulted in a substantial increase in rapamycin biosynthesis confirming their positive regulatory role, while deletion of both from the chromosome of S. hygroscopicus resulted in a complete loss of antibiotic production. Complementation studies indicated an essential role of the RapG regulator for rapamycin biosynthesis and a supportive role of RapH. A direct effect of rapH and rapG gene products on the promoter of the rapamycin polyketide synthase operon, rapA-rapB, was observed using the chalcone synthase gene rppA as a reporter system.