Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

Adam Thomas,William Finnigan,Mark Van Der Giezen,Nicholas Harmer,Rhys Cutlan

doi:10.1038/s42003-019-0677-y

Adam Thomas, William Finnigan + Show 3 more

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https://doi.org/10.1038/s42003-019-0677-y

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Journal: Communications Biology	Publication Date: Nov 22, 2019
Citations: 38	License type: open-access

Affiliation: University of Exeter

Abstract

Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.

Highlights

Carboxylic acid reductases (CARs) are biocatalysts of industrial importance
We previously reported that well characterized extant CARs (ExCARs) are barely suitable for reactions above 37 °C
We previously reported a dataset of 124 CAR homologs identified from the CAR1 family[10]

Summary

Introduction

Carboxylic acid reductases (CARs) are biocatalysts of industrial importance Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Carboxylic acid reductases (CARs; E.C. 1.2.1.30) are a family of enzymes with increasing relevance to green chemistry. They catalyze the reduction of an aliphatic or aromatic acid to the respective aldehyde, using ATP and NADPH as cofactors[10,11]. This reaction is otherwise challenging to achieve chemically or biochemically. CAR subgroup I can be further split into five families, of which family

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