Recombinant strains for the enhanced production of bioengineered rapalogs

• Published in: Metabolic engineering Vol. 15; pp. 167 - 173
• Main Authors: Kendrew, Steven G., Petkovic, Hrvoje, Gaisser, Sabine, Ready, Sarah J., Gregory, Matthew A., Coates, Nigel J., Nur-e-Alam, Mohammad, Warneck, Tony, Suthar, Dipen, Foster, Teresa A., McDonald, Leonard, Schlingman, Gerhard, Koehn, Frank E., Skotnicki, Jerauld S., Carter, Guy T., Moss, Steven J., Zhang, Ming-Qiang, Martin, Christine J., Sheridan, Rose M., Wilkinson, Barrie
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.01.2013
• Subjects: Bioengineering; Clinical trials; Genetic Enhancement - methods; Mutagenesis, Site-Directed - methods; Polyketide; Rapamycin; Recombination, Genetic - genetics; Sirolimus - isolation & purification; Sirolimus - metabolism; Species Specificity; Streptomyces; Streptomyces - classification; Streptomyces - physiology; Polyketide; Bioengineering; Rapamycin
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2012.11.001

The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials. ► Combined genetic and mutational approach yields efficient rapalog production strain. ► Deletion of rapK encoding the chorismatase required for starter unit biosynthesis. ► Mutasynthesis of hard to access rapalogs enabled. ► Ectopic expression enhances congener profile of bioengineered rapalogs.