Fu Bing, Chen Meng, Bao Xianfeng, Lu Jiajie, Zhu Zhiwen, Guan Fuyao, Yan Chuyang, Wang Peize, Fu Linglin, Yu Ping
College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, Zhejiang Province, 310035, People's Republic of China.
College of Forestry Science and Technology, Lishui Vocational and Technical College, 357 Zhongshan Street North, Lishui, Zhejiang Province, 323000, People's Republic of China.
Synth Syst Biotechnol. 2024 Apr 16;9(3):503-512. doi: 10.1016/j.synbio.2024.04.011. eCollection 2024 Sep.
Vitamin B is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant BL21.
维生素B是一种必需的水溶性维生素。对于大多数原核生物来说，一种名为黄素腺嘌呤二核苷酸合酶的双功能酶催化核黄素依次转化为黄素单核苷酸和黄素腺嘌呤二核苷酸。在本研究中，将含有核黄素合成六个关键基因的质粒pNEW-AZ转化到缺失黄素单核苷酸核糖开关的R2菌株中，得到R5菌株。R5菌株可产生540.23±5.40mg/L核黄素，比含有六个关键基因的质粒pET-AE和pAC-Z的R4菌株高10.61%。为了进一步提高核黄素的产量，进行了同源匹配和分子对接以鉴定黄素腺嘌呤二核苷酸合酶的关键氨基酸残基。确定了九个点突变位点。通过比较核黄素激酶活性，选择与野生型黄素腺嘌呤二核苷酸合酶相比分别降低29.90%和89.32%的T203D和N210D突变进行基因组的CRISPR/Cas9基因编辑，产生工程菌株R203和R210。将pNEW-AZ转化到R203中，得到R6。R6产生657.38±47.48mg/L核黄素，比R5增加了21.69%。本研究有助于重组BL21中核黄素的高产。
