Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv, 79005, Ukraine.
J Ind Microbiol Biotechnol. 2014 May;41(5):823-35. doi: 10.1007/s10295-014-1422-7. Epub 2014 Mar 5.
Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett-Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH2PO4, glycine, and (NH4)2SO4. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.
黄素以黄素单核苷酸 (FMN) 和黄素腺嘌呤二核苷酸 (FAD) 的形式在代谢中发挥重要作用，作为氧化还原酶和其他酶的辅因子。黄素核苷酸在食品工业和医药中有应用；FAD 补充剂已被有效地用于治疗某些遗传性疾病。FAD 是通过生物技术生产的；然而，这种化合物比核黄素贵得多。产黄素酵母 Candida famata 从 FMN 和 ATP 合成 FAD，该反应由 FAD 合成酶催化，该酶是 FAD1 基因的产物。使用强组成型启动子 TEF1 表达 FAD1 导致 FAD 合成酶活性、FAD 过量产生和分泌到培养基中增加 7-15 倍。对其中一株重组菌株 C. famata T-FD-FM 27 在不同生长条件下的 FAD 生产效率进行了评估。首先，进行了两水平 Plackett-Burman 设计，以筛选对 FAD 生产有显著影响的培养基成分。其次，采用中心复合设计研究了所选因素的最佳值，以实现最大 FAD 产量。FAD 产量对腺嘌呤、KH2PO4、甘氨酸和(NH4)2SO4 的浓度变化最为显著。与非优化对照条件相比，实施这些优化策略可使 FAD 产量增加 65 倍。在优化条件下培养 40 小时的重组菌株可实现 451mg/L 的 FAD 积累。因此，首次获得了过量生产 FAD 的酵母菌株，并设计了用于该核苷酸最大生产的生长培养基组成。
