Dmytruk K V, Abbas C A, Voronovsky A Y, Kshanovska B V, Sybirna K A, Sybirny A A
Ukr Biokhim Zh (1999). 2004 Jan-Feb;76(1):78-87.
The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.
通过常规筛选方法分离得到的产核黄素酵母法夫酵母（Candida famata）核黄素高产突变体用于工业生产维生素B2。最近，利用亮氨酸缺陷型（leu2）突变体作为受体菌株，酿酒酵母（Saccharomyces cerevislae）的LEU2基因作为选择标记，开发了一种法夫酵母的转化系统。本文描述了基于已开发的该酵母转化系统克隆法夫酵母核黄素合成基因的过程。从先前筛选的法夫酵母leu2菌株中分离出核黄素自养突变体。构建了法夫酵母基因组DNA文库，并通过在不含亮氨酸和核黄素的培养基上互补生长缺陷来克隆核黄素合成的相应结构基因。结果，分离出了携带编码GTP环化水解酶、还原酶、二甲基核醇基蝶啶合酶、磷酸二羟基丁酮合酶和核黄素合酶的RIB1、RIB2、RIB5、RIB6和RIB7基因的DNA片段，并随后将其亚克隆到尽可能小的片段。含有这些基因的质粒成功地互补了另一种产黄素酵母季也蒙毕赤酵母（Pichia guilliermondii）相应突变体的核黄素营养缺陷型。这表明克隆的是法夫酵母核黄素合成的结构基因而非一些抑制基因。
