Roles of rapH and rapG in Positive Regulation of Rapamycin Biosynthesis in Streptomyces hygroscopicus

Abstract

Rapamycin is an important macrocyclic polyketide produced by Streptomyces hygroscopicus and 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, the products of genes rapH and rapG showed high similarities with two different families of transcriptional activators, LAL and AraC, respectively. Overexpression 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.

A key developmental regulator controls the synthesis of the antibiotic erythromycin in Saccharopolyspora erythraea

“…Together, these data suggest that BldD positively regulates the ery genes. However, because the mutation left some synthesis of erythromycin intact, it differs from deletions of activators in many streptomycetes that abolish the production of an antibiotic completely (3,23). Our observations more closely resemble the case of S. noursei, in which a strain with a deletion of a nystatin synthesis regulator still produced some antibiotic (24).…”

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…60 Recently, the Biotica group showed that RapH and RapG have important roles as positive regulators in rapamycin biosynthesis. 23 Overexpression of rapH and rapG under the control of the ActII-ORF4/PactI activator/promoter in S. hygroscopicus enhanced the production of rapamycin by approximately 27-55% and 20-32%, respectively. The ActII-ORF4/PactI expression system, which is a robust activator/promoter expression system that has been widely used for many actinomycetes, including S. hygroscopicus, was used to minimize the potential self-regulatory interference of RapH and/or RapG and other endogenous regulatory genes.…”

“…For example, the Biotica group (Cambridge, UK) revealed various positive regulatory elements in rapamycin biosynthesis, 23 the Wyeth group (Madison, NJ, USA) modified the pipecolate ring of rapamycin and Leadlay and coworkers 24 synthesized several rapamycin analogs with modifications to the cyclohexyl moiety through genetic engineering and mutasynthesis. Recently, Graziani 25 provided a review article on the advances in the field of rapamycin biosynthesis and neuroprotective activity of rapamycin analogs.…”

“…Subsequent complementation with rapH and rapG resulted in a restoration of antibiotic production, underlying their essential role. 23 …”

Discovering Metabolic Products of Cryptic Biosynthetic Pathways

“…This cluster is not associated with any metabolite production and is found to be poorly expressed. These workers expressed this cryptic gene cluster using large ATP-binding regulator of the LuxR family (LAL) protein, the members of which are known to be activator of pikromycin [3] and rapamycin [4]. NMR analysis of the expressed product suggested that the product is a novel macrolide which was named stambomycin.…”