The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

Abstract

AbstractIn this work, we introduce the application of proton transfer reaction mass spectrometry (PTR‐MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC‐MS)‐based methods, PTR‐MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR‐MS‐based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR‐Qi‐ToF‐MS were compared with GC‐MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR‐Qi‐ToF‐MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC‐MS analysis. This study provides a first basis for the application of rapid PTR‐Qi‐ToF‐MS detection, in combination with multi‐well formats, in HTS screening methods for the selection of highly productive terpene synthases.