Abstract
The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.
Highlights
Biocatalysis provides an alternative and increasingly attractive sustainable pathway for the production of high-value chemical building blocks and intermediates
Applying desorption electrospray ionization (DESI) MS to crude reaction mixtures permits the analysis of products in situ[2] (Figure 1), which has significant benefits for monitoring biocatalytic reactions compared to more traditional chromatographic MS methods
Coupling the direct infusion of biotransformations to the mass spectrometer (DiBT-MS) high throughput screening method, which provides semiquantitative chemical information, to a protein engineering strategy accelerates the evolution of enzymes as demonstrated here
Summary
Biocatalysis provides an alternative and increasingly attractive sustainable pathway for the production of high-value chemical building blocks and intermediates. Applying desorption electrospray ionization (DESI) MS to crude reaction mixtures permits the analysis of products in situ[2] (Figure 1), which has significant benefits for monitoring biocatalytic reactions compared to more traditional chromatographic MS methods. This approach shortens the analysis time and removes the need for much of the solvent. The ability to correlate the spatial positions of spotted reactions and colonies with products provides a visual reference on the identified enzyme “hits”, followed by DNA extraction/PCR amplification to determine the sequence.[3] Coupling the direct infusion of biotransformations to the mass spectrometer (DiBT-MS) high throughput screening method, which provides semiquantitative chemical information, to a protein engineering strategy accelerates the evolution of enzymes as demonstrated here
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