Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.

BackgroundErythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement.ResultsWe used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis.ConclusionsSACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

Potato production worldwide is threatened by late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary. Highly resistant potato cultivars were developed in breeding programs, using resistance gene pyramiding methods. In Sárpo Mira potatoes, five resistance genes (R3a, R3b, R4, Rpi-Smira1, and Rpi-Smira2/R8) are reported, with the latter gene assumed to be the major contributor. To study the level of late blight resistance conferred by the Rpi-Smira2/R8 gene, potato genotypes with only the Rpi-Smira2/R8 gene were selected from progeny population in which susceptible cultivars were crossed with Sárpo Mira. Ten R8 potato genotypes were obtained using stepwise marker-assisted selection, and agroinfiltration of the avirulence effector gene Avr4. Nine of these R8 genotypes were infected with both Slovenian P. infestans isolates and aggressive foreign isolates. All the progeny R8 genotypes are resistant to the Slovenian P. infestans isolate 02_07, and several show milder late blight symptoms than the corresponding susceptible parent after inoculation with other isolates. When inoculated with foreign P. infestans isolates, the genotype C571 shows intermediate resistance, similar to that of Sárpo Mira. These results suggest that Rpi-Smira2/R8 contributes to late blight resistance, although this resistance is not guaranteed solely by the presence of the R8 in the genome.

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