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A secretion‐based dual fluorescence assay for high‐throughput screening of alcohol dehydrogenases
Author(s) -
Lu Hongyuan,
Yu Shiqin,
Qin Fengyu,
Ning Wenbo,
Ma Xiaoqiang,
Tian Kaiyuan,
Li Zhi,
Zhou Kang
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27677
Subject(s) - cofactor , fluorescence , high throughput screening , biochemistry , secretion , chemistry , alcohol , enzyme , green fluorescent protein , chromatography , biology , combinatorial chemistry , gene , physics , quantum mechanics
Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion‐based dual fluorescence assay (SDFA) for high‐throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197‐fold higher k cat / k m value toward ( S )‐2‐octanol compared to its wild type.