Dumina M V, Zhgun A A, Kerpichnikov I V, Domracheva A G, Novak M I, Valiakhmetov A Ia, Knorre D A, Severin F F, Él'darov M A, Bartoshevich Iu É
Prikl Biokhim Mikrobiol. 2013 Jul-Aug;49(4):372-81. doi: 10.7868/s0555109913040041.
Vectors for the expression of the CefT transporter of the MFS family in 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 created. 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, tpo 1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-yield strain VKM F-4081D of A. chrysogenum, expressing the cefT-cfp fusion, was obtained by an agrobacteria conjugated transfer. 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 finite product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediators.
已构建用于在产β-内酰胺抗生素头孢菌素C的产黄青霉以及酿酒酵母中表达MFS家族的CefT转运蛋白的载体，该载体与青色荧光蛋白（CFP）融合。研究了酵母细胞中CefT-CFP杂交蛋白的亚细胞定位。结果表明，CefT-CFP杂交蛋白能够互补酿酒酵母中编码直系同源MFS转运蛋白的qdr3、tpo 1和tpo3基因，使相应菌株对亚精胺、溴化乙锭和潮霉素B具有抗性。通过农杆菌共轭转移获得了表达ceft-cfp融合蛋白的产黄青霉高产菌株VKM F-4081D。还表明，产黄青霉VKM F-4081D中ceft的组成型表达导致头孢菌素C及其前体的生物合成谱发生变化。这导致培养液中积累的最终产物减少25-35%，同时其中间体浓度增加。
