title: A fast and sensitive coupled enzyme assay for the measurement of l-threonine and application to high-throughput screening of threonine-overproducing strains.
abstract: Amino acids are important fermentation products that are used in the food, animal feed, pharmaceutical, and cosmetics industries. Development of a better quantitation assay to measure amino acid levels will facilitate research and application in medical and industrial fields. Here we report the development of an assay that allows the rapid measurement of threonine concentration. l-threonine aldolase (LTA) catalyzes the cleavage of threonine to glycine and acetaldehyde. The resulting acetaldehyde is further converted into ethanol by alcohol dehydrogenase accompanying NADH reduction. Therefore, monitoring the changes in NADH concentration can serve as a readout for the amount of threonine present within certain range. This LTA assay can distinguish a 0.5mM difference in threonine concentration in some certain mediums. We successfully utilized this assay to measure l-threonine levels in industrial fermentation samples. Moreover, we were able to scale up this assay to 96-well format and use it in high-throughput screening of threonine-overproducing strains. We show this assay is simple, accurate, specific and suitable for determination of threonine concentration in large numbers of samples.
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Yongfei
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Key Laboratory of Structure and Performance for Functional Molecule, College of Chemistry, Tianjin Normal University, Tianjin 300387, China.
authors.forename: Feiran
authors.initials: F
authors.lastname: Li
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Cao
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Tianjin Key Laboratory of Structure and Performance for Functional Molecule, College of Chemistry, Tianjin Normal University, Tianjin 300387, China.
authors.forename: Yuxiu
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zheng
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; National Engineering Laboratory for Industrial Enzymes, Tianjin 300308, China. Electronic address: zhang_dw@tib.cas.cn.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhang
mesh_terms: D000821:Animal Feed / Q000032:analysis; D000818:Animals; D005285:Fermentation; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000706:statistics & numerical data; D007218:Industrial Microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000032:analysis* / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D013912:Threonine; D012696:Glycine Hydroxymethyltransferase
keywords: Coupled enzyme assay; High throughput screening; Threonine determination; l-Threonine aldolase