Mayerl F, Piret J, Kiener A, Walsh C T, Yasui A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
J Bacteriol. 1990 Oct;172(10):6061-5. doi: 10.1128/jb.172.10.6061-6065.1990.
The gene encoding Anacystis nidulans 5-deazaflavin-dependent photolyase (phr) was inserted into the Streptomyces vector pIJ385 to form a transcriptional fusion with the neomycin resistance (aph) gene. The resulting plasmid, pANPL, was introduced into Streptomyces coelicolor, a host which exhibits no detectable photolyase activity and provides 5-deazaflavins. Transformants expressed functional photolyase and could be cultured at much higher cell densities than A. nidulans. A two-step affinity protocol was used to purify photolyase to homogeneity. High-pressure liquid chromatographic analysis established the presence of 5-deazaflavin cofactors in the enzyme, showing that this expression system allows heterologous production of 5-deazaflavin-class photolyases.
将编码集胞藻5-脱氮黄素依赖性光裂合酶（phr）的基因插入链霉菌载体pIJ385中，与新霉素抗性（aph）基因形成转录融合。所得质粒pANPL被导入天蓝色链霉菌，该宿主没有可检测到的光裂合酶活性，但能提供5-脱氮黄素。转化体表达功能性光裂合酶，并且可以在比集胞藻更高的细胞密度下培养。采用两步亲和方案将光裂合酶纯化至同质。高压液相色谱分析确定该酶中存在5-脱氮黄素辅因子，表明该表达系统能够异源生产5-脱氮黄素类光裂合酶。
