Hagihara T, Fujio T, Aisaka K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Japan.
Appl Microbiol Biotechnol. 1995 Jan;42(5):724-9. doi: 10.1007/BF00171952.
The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 x 10(3)-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. ammoniagenes. The FAD-synthetase-gene-amplified C. ammoniagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn2+ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyl-transferase activity and resulted in the accumulation of FMN.
尝试通过与对应于N端氨基酸序列的合成DNA杂交，从产氨棒杆菌中克隆一个具有黄素激酶和FMN腺苷酸转移酶活性的双功能FAD合成酶基因。克隆得到的经PstI酶切的4.4×10³碱基（4.4 kb）片段在大肠杆菌中不能表达FAD合成酶活性，但在产氨棒杆菌中能使这两种活性提高约20倍。将FAD合成酶基因扩增的产氨棒杆菌细胞用于从FMN或核黄素生产FAD。与亲本菌株相比，从FMN生产FAD的产量提高了四到五倍，摩尔产率达到90%。从核黄素生产FAD的产量提高了约八倍，摩尔产率为50%。在从核黄素转化为FAD的反应混合物中添加Zn²⁺会特异性抑制腺苷酸转移酶活性，导致FMN积累。
