Abstract
Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.
Highlights
Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; engineering them to have novel or enhanced activity is limited by technologies for sensing product formation
We combine this approach with printed droplet microfluidics (PDM) to prepare, print, and screen all mutants from a semi-rationally varied fourposition library of the Gerbera hybrida G2PS1 type-3 polyketide synthase[8], comprising 1960 codon-shuffled members (Fig. 2a)
This enzyme is responsible for the biosynthesis of triacetic acid lactone (TAL) through condensation of a starter acetyl-CoA unit with two malonyl-CoA molecules and subsequent cyclization of the triketide chain[8,9]
Summary
Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. We describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis. 1234567890():,; Enzyme engineering uses an iterative cycle in which libraries of gene variants are designed, synthesized into proteins, and tested for the activity of interest[1]. The success of these engineering campaigns, is dependent on technologies for conducting these steps. MALDI MS ion imaging m/z x y approach provides a general way to map the catalysis of a mutated enzyme, to characterize the range of products it generates, and to recover the sequences of variants with desired activities
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