Direct evolution of riboflavin kinase significantly enhance flavin mononucleotide synthesis by design and optimization of flavin mononucleotide riboswitch

被引：6

作者：

Du, Yuxuan ^{[1
]}

Zhang, Xinyi ^{[1
]}

Zhang, Hengwei ^{[1
]}

Zhu, Rongshuai ^{[1
]}

Zhao, Zhenqiang ^{[1
]}

Han, Jin ^{[1
]}

Zhang, Di ^{[1
]}

Zhang, Xiaoling ^{[1
]}

Zhang, Xian ^{[1
]}

Pan, Xuewei ^{[1
]}

You, Jiajia ^{[1
]}

Rao, Zhiming ^{[1
,2
]}

机构：

[1] Jiangnan Univ, Sch Biotechnol, Key Lab Ind Biotechnol, Minist Educ, Wuxi 214122, Jiangsu, Peoples R China

[2] Jiangnan Univ, Sch Biotechnol, Key Lab Ind Biotechnol, Lab Appl Microorganisms & Metab Engn,Minist Educ, Wuxi 214122, Peoples R China

来源：

BIORESOURCE TECHNOLOGY | 2023年 / 381卷

基金：

中国国家自然科学基金; 中国博士后科学基金;

关键词：

Bifunctional riboflavin kinase; FMN; adenylyltransferase; Base editor; High-throughput screening; Biosensors; flavin mononucleotide (FMN); BINDING; TRANSCRIPTION;

D O I：

10.1016/j.biortech.2023.128774

中图分类号：

S2 [农业工程];

学科分类号：

0828 ;

摘要：

Flavin mononucleotide (FMN) is the active form of riboflavin. It has a wide range of application scenarios in the pharmaceutical and food additives. However, there are limitations in selecting generic high-throughput screening platforms that improve the properties of enzymes. First, the biosensor in response to FMN concen-tration was constructed using the FMN riboswitch and confirmed the function of this sensor. Next, the FMN binding site of the sensor was saturated with a mutation that increased its fluorescence range by approximately 127%. Then, the biosensor and the base editing system based on T7RNAP were combined to construct a platform for rapid mutation and screening of riboflavin kinase gene ribC mutants. The mutants screened using this plat-form increased the yield of FMN by 8-fold. These results indicate that the high-throughput screening platform can rapidly and effectively improve the activity of target enzymes, and provide a new route for screening industrial enzymes.

引用

页数：9

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