Site-directed mutagenesis of bifunctional riboflavin kinase/FMN adenylyltransferase via CRISPR/Cas9 to enhance riboflavin production  

作　　者：Bing Fu Meng Chen Xianfeng Bao Jiajie Lu Zhiwen Zhu Fuyao Guan Chuyang Yan Peize Wang Linglin Fu Ping Yu 

机构地区：[1]College of Food Science and Biotechnology,Zhejiang Gongshang University,149 Jiaogong Road,Hangzhou,Zhejiang Province,310035,People’s Republic of China [2]College of Forestry Science and Technology,Lishui Vocational and Technical College,357 Zhongshan Street North,Lishui,Zhejiang Province,323000,People’s Republic of China [3]Lishui Institute for Quality Inspection and Testing,395 Zhongshan Street,Lishui,Zhejiang Province,323000,People’s Republic of China

出　　处：《Synthetic and Systems Biotechnology》2024年第3期503-512,共10页合成和系统生物技术（英文）

基　　金：the Natural Science Foundation of Zhejiang Province,China(No.LY21C200006)。

摘　　要：Vitamin B_(2)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 E.coli BL21.

关 键 词：Vitamin B_(2) FAD synthase Escherichia coli BL21 Riboflavin kinase CRISPR/Cas9 

分 类 号：Q93[生物学—微生物学]