Practical high-throughput screening assays to engineer the substrate scope of transketolase variants.

Transketolases are thiamine diphosphate-dependent enzymes widely studied in biocatalysis for their ability to stereoselectively form acyloins by carbon-carbon bond coupling. In this chapter, we describe two high-throughput assay approaches for engineering transketolase towards non-natural substrate acceptance. First, we present a pH-sensitive method in solution that induces CO₂ release from cleavage of a keto acid as donor substrate. Inexpensive pH indicators (phenol red or HPTS to be measured by absorption or fluorescence changes, respectively) display these changes as an indirect readout of substrate consumption, enabling rapid screening across variant libraries. This method is simple to perform and broadly applicable, but it yields only an indirect signal of catalytic conversion and is sensitive to experimental variability. Next, we introduce a hydroxamate assay that directly monitors product formation colorimetrically via iron(III) chelation. The resulting dark-red complex indicates successful synthesis of an N-aryl hydroxamic acid, thereby improving specificity and reducing background interference. This assay is compatible with different formats, either in liquid media on microtiter plates or by colony screening on solid phase, the latter offering significant time and resource savings by detecting active variants on membrane copies of agar growth plates. Together, these assays provide a powerful and versatile toolkit for directed evolution and enzyme mining of TKs, ultimately accelerating the discovery of robust variants suited for industrial biocatalysis.

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