Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

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.

A Reconstructed Common Ancestor of the Fatty Acid Photo‐decarboxylase Clade Shows Photo‐decarboxylation Activity and Increased Thermostability

“…[22,23] Reconstructed enzymes may not only have a broader substrate range compared to their modern descendants, [24] but often show improved thermostability and solvent tolerance. [25,26] Therefore, we hypothesized that the common ancestor in the FAP clade identified by Sorigue et al [7] would still show photo-decarboxylation but with improved stability and/or soluble production in E. coli as demonstrated previously. [27,28] To verify this, we set out to reconstruct three possible ancestors of this clade and investigate their photo-decarboxylative activity.…”

“…It has emerged as a proficient method to produce functional proteins that keep the aspects of the protein family [22,23] . Reconstructed enzymes may not only have a broader substrate range compared to their modern descendants, [24] but often show improved thermostability and solvent tolerance [25,26] . Therefore, we hypothesized that the common ancestor in the FAP clade identified by Sorigue et al [7] .…”

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

“…This EcPPTase was used as the co-expressed PPTase for activation of apo-CARs in this and other studies. [18,34,37,54,55] To co-express a PPTase that binds and activates the PCP-domain from MmCAR perhaps more efficiently than EcPPTase, the endogenous PPTases from known CAR origins (Nocardia iowensis, Neurospora crassa, Mycobacterium marinum) as well as SfpPPTase from Bacillus subtilis, which was widely used in CAR research, [12,27,56,57] were cloned and coexpressed with MmCAR. Biphasic whole-cell bioconversions of 3a were carried out to study the effect.…”

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

“…The design method consists of three steps: (1) inference of an ancestral sequence based on a comparison of homologous amino acid sequences; (2) artificial synthesis of a gene encoding the inferred amino acid sequence; and (3) expressing the gene in a host organism such as Escherichia coli . Proteins reconstructed in this way are often highly thermostable 29 – 32 . Moreover, by substituting putative ancestral amino acids into natural enzymes, the thermostability of a natural protein is highly likely to be increased without compromising the catalytic activity 33 – 37 .…”