Fakultät für Naturwissenschaften, Brandenburgische Technische Universität Cottbus-Senftenberg, Großenhainer Str. 57, 01968, Senftenberg, Germany,
Appl Microbiol Biotechnol. 2013 Dec;97(23):10143-53. doi: 10.1007/s00253-013-5266-2. Epub 2013 Oct 4.
The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60% of the hyphal cells. For the remaining 40%, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.
半子囊菌棉阿舒囊霉会形成菌丝体。营养压力会导致其分化为孢子囊。这些孢子囊会产生孢子。与此同时，黄色色素核黄素也会生成。细胞内积累的核黄素，以明亮的绿色荧光显现，只在 60%的菌丝细胞中观察到。对于其余的 40%，尚不清楚这些细胞是简单地将核黄素输出，还是其生物合成与积累的细胞相比仍然下调。本工作中采用的方法是通过细胞壁的酶降解将菌丝体转化为原生质体。然后，根据核黄素积累，通过荧光激活细胞分选对原生质体进行分选。当使用表达 lacZ 的报告菌株，该菌株受核黄素生物合成中最重要的启动子 RIB3 的控制时，发现绿色原生质体的报告基因活性比透明原生质体高 10 多倍。这一结论基于总蛋白和己糖激酶比活性，后者是组成型表达的标志物。这些结果表明，棉阿舒囊霉的菌丝体细胞在核黄素过量产生和积累方面表现出不同的表型。
