Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae |
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Authors: | M V Dumina A A Zhgun I V Kerpichnikov A G Domracheva M I Novak A Ya Valiachmetov D A Knorre F F Severin M A Eldarov Yu E Bartoshevich |
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Institution: | 16508. Bioengineering Center, Russian Academy of Sciences, Moscow, 117312, Russia 26508. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, 142290, Russia 36508. Belozersky Institute of Physicochemical Biology, Moscow State University, Moscow, 119899, Russia
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Abstract: | Vectors for the expression of MFS transporter CefT of Acremonium chrysogenum—a producer of beta-lactam antibiotic cephalosporin C—and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been generated. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-producing strain A. chrysogenum VKM F 4081D, expressing the cefT-cfp fusion construct, was obtained by an agrobacteria mediated transformation. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25–35% decrease in the amount of the final product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediates. |
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