A secretion-based dual fluorescence assay for high-throughput screening
of alcohol dehydrogenases
Abstract
Alcohol dehydrogenases (ADHs) play key roles in the production of
various chemical precursors that are essential in pharmaceutical and
fine chemical industries. To achieve 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 fluorescence protein (MsfGFP), which could result in
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 activity of the ADH based on a red fluorescence signal,
whose increase is coupled to formation of NADH (a redox co-factor 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 kcat/km value toward (S)-2-octanol
compared to its wild-type.