Eliasson Lantz, Anna

Mijakovic, Ivan

Sohoni, Sujata Vijay

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 17, 2017Synthetic Promoter Library for modulation of actinorhodin production in StreptomycescoelicolorA3(2)Sohoni, Sujata Vijay; Mijakovic, Ivan; Eliasson Lantz, AnnaPublication date:2009Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Sohoni, S. V., Mijakovic, I., & Eliasson Lantz, A. (2009). Synthetic Promoter Library for modulation ofactinorhodin production in Streptomyces coelicolorA3(2). Poster session presented at 15th Internationalsymposium on the biology of actinomycetes, Shanghai, China, .Synthetic Promoter Library for modulation of  actinorhodin production in Streptomyces coelicolor A3(2)1Center for Microbial Biotechnology, Department of Systems Biology, Denmark Technical University, 2800 Kgs. Lyngby DENMARK.2Microbiologie et Génétique Moléculaire, AgroParisTech-INRA-CNRS, Route de Thiverval, F-78850 Thiverval-Grignon FRANCEEmail: svs@bio.dtu.dk, Ivan.Mijakovic@grignon.inra.fr, ael@bio.dtu.dkSujata Vijay Sohoni1, Ivan Mijakovic1,2, Anna E. Lantz1Introduction:Genetic manipulation tools have been successfully used for improving properties of industrial microorganisms. Most of these approaches involveover-expression of a gene for the rate limiting enzyme or deletion of a gene situated at a branching point in case of branched pathways. Thesesimplistic all-or-nothing approaches have been fruitful in some cases, but can fall short when careful optimization of gene expression is needed inorder to tune the modified pathway with the rest of the cellular metabolism. Promoter strength plays an important role in the resulting levels of geneexpression. The synthetic promoter technology, based on randomization of the promoter sequences, has been successfully employed to constructpromoter libraries in order to optimize levels of gene expression.Synthetic promoter technology is based on the fact that the spacer sequences surrounding the consensus -35 and -10 regions of bacterial promoterscontribute significantly to promoter strength. Randomizing these spacer sequences results in Synthetic Promoter Libraries.In the current study the native promoter of actII orf4 was modified by randomizing spacer sequence between -35 box and -10 box and 5 nucleotidebefore and 5 nucleotides after -35 box and -10 box respectively. The resulting library was screened and characterized for production of actinorhodin.Screening of  Synthetic Promoter Library•Around 10,000 colonies were screened by visual screening•200 colonies having a blue actinorhodin hallow were selected for characterization•Out of  the 200 colonies,12 colonies were subjected to detailedphysiology studiesCharacterization of  Synthetic Promoter LibraryConstruction of  Synthetic promoter LibraryScreening of  the library by image analysisat -20°CResults and DiscussionReferences(1) Solem, C., Jensen, P. R. 2002. Modulation of gene expression made easy. Appl.Environ.Microbiol.,68, 5, 2397-2403(2) Arias, P., Fernandez-Moreno, M. A., Malpartida, F. 1999. Characterization of the pathway-specificpositive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as aDNA-binding protein. Journal of Bacteriology, 181(22), 6958-6968AcknowledgementsAuthors would like to thank Prof. Gunther Muth (University of Tubingen) forproviding plasmid pGM160.Authors would also like to thank Dr. Prashant Bapat and Dr. Kiran Patil forproviding algorithm for the image analysisSome of  the SPL colonies-20°COutput of  image analysis showing onset of  antibiotics UDP (red) and Act (Blue)Representation of  the library based  on  yields of  actinorhodin on biomass°CSPL 20 strain in reactor20°CA promoter library producing different yields of  actinorhodin was constructed. SPL 20 was found to be very interesting and hence studied in details in bioreactor.12 strains showing higher and lower yields than that of  the wild type strain were also studied in more details and the promoter sequnces are being obtained.SPL 20 is a very good example of  the principle of  promoter library. Hence the library was successfully constructed. The promoters thus obtained could be generalized for various expression levels of  different genes according to the requirements

Synthetic Promoter Library for modulation of actinorhodin production in Streptomyces coelicolorA3(2)

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Synthetic Promoter Library for modulation of actinorhodin production in Streptomyces coelicolorA3(2)

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