High-throughput Discovery Lab

The application of enzymes to catalyze industrial reactions, known as “biocatalysis”, has the potential to make chemical processes less contaminant and more sustainable, following the principles of Green Chemistry.  In fact, the enzymatic synthesis of nucleoside analogue-based drugs (NABDs) offers several advantages over chemical methods, e.g.: i) one-pot reactions; ii) higher regio- and stereo-selectivity; iii) higher yields and product purities; and iv) eco-friendly technology.  The most habitual strategy for the enzymatic synthesis of NABDs is the transglycosylation from a nucleoside donor to a nucleobase acceptor. In this respect, several enzymes, such as nucleoside 2′-deoxyribosyltransferases (NDTs) or nucleoside phosphorylases (NPs) have been used as biocatalysts for the one-pot, tailor-made synthesis of NABDs.

The general objective of the project is to develop an evolved NDT for the synthesis of high added-value NABDs that exhibits a high activity towards modified C2’ nucleoside analogues, such as 2’-fluoro-2’deoxypurine nucleosides or arabinose-containing nucleoside analogues. To that end, the Leishmania mexicana NDT has been reported to accept 2’F-dG, 2’F-dI, araG and araH as substrates, however at long reaction times and high enzyme loading.  This is clearly insufficient for industrial application.  In this work, a biological selection will be used based on a guanine auxotroph to achieve variants that are a good starting point for directed evolution using droplet microfluidics

This general objective is divided in the following sub-objectives:

• Setup of a selection against 2’F-dG and araG using an auxotrophic strain for guanine
• Generation of randomized libraries of Leishmania mexicana NDT (LmeNDT) by epPCR
• Selection of LmeNDT variants against against 2’F-dG and araG
• Confirmation of hits using a fluorimetric assay in multiwell-plates