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. 2007 Jul;189(13):4756-63.
doi: 10.1128/JB.00129-07. Epub 2007 Apr 27.

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

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Free PMC article

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

Enej Kuscer et al. J Bacteriol. .
Free PMC article

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.

Figures

FIG. 1.
FIG. 1.
Rapamycin biosynthetic gene cluster, showing the location and the area of deletion of the rapH and rapG genes. Promoter PrapA, which controls the main PKS operon rapA-rapB, is also shown. To the right side of the rapA-rapB operon lie genes encoding RapP, a pipecolate-incorporating enzyme, and RapC, which together with RapA and RapB represent the rapamycin PKS.
FIG. 2.
FIG. 2.
Variability of rapamycin yields in S. hygroscopicus strains within a single fermentation run. **, data for WT:HG and ΔHG:HG were obtained from a separate fermentation run and were normalized against the wild-type control (WT). Bars encompass 95% of the sample population, with asterisks representing the mean values, horizontal line representing the median values, and perpendicular lines indicating outliers and extreme values. The data were analyzed using the SAS/STAT program. np, genes under control of the native promoters.
FIG. 3.
FIG. 3.
High-pressure liquid chromatography analysis of rapamycin (retention time, ca. 8.9 to 9.0 min). Wild-type (w.t.), rapH overexpression (w.t.:rapH), rapH rapG deletion (ΔHG), and partial (ΔHG:rapG) and full (ΔHG:rapHG-np) complementation strains are shown. All rapamycin peaks were verified against a rapamycin standard.
FIG. 4.
FIG. 4.
Chalcone synthase (CHS) reporter gene expression. The RppA-PrapA plasmid (A) contains the PrapA promoter in front of the rppA gene. Error bars indicate standard deviations.

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