Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery

Author:

Rizzo Andrea1,Aranda Carmen1ORCID,Galman James1,Alcasabas Annette1,Pandya Akash1,Bornadel Amin1,Costa Bruna1,Hailes Helen C.2,Ward John M.3,Jeffries Jack W. E.3,Dominguez Beatriz1

Affiliation:

1. Johnson Matthey Unit 260, Cambridge Science Park Cambridge CB4 0PZ

2. Department of Chemistry University College London 20 Gordon Street London WC1H 0AK UK

3. Department of Biochemical Engineering University College London Gower Street, Bernard Katz Building London WC1E 6BT UK

Abstract

AbstractBio‐processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose‐5‐phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence‐based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA‐61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA‐61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non‐natural substrates, compared to well‐known DERAs.

Publisher

Wiley

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