Exploring the abundance of oleate hydratases in the genus Rhodococcus\u2014discovery of novel enzymes with complementary substrate scope

Hanna Busch,Jean-Marc Daran,Marcel Van Den Broek,Natália Alvarenga,Simona Lu,Peter-Leon Hagedoorn,Ulf Hanefeld,Fabio Tonin

doi:10.1007/s00253-020-10627-7

Hanna Busch, Jean-Marc Daran + Show 6 more

Open Access

https://doi.org/10.1007/s00253-020-10627-7

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Abstract

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.

Highlights

Rhodococcus is a genus of aerobic, gram-positive bacteria and is known for its diverse biocatalytic activity towards a plethora of substrates
In the course of identifying novel hydratases with interesting properties in the genus Rhodococcus, an Orthologous Matrix (OMA) algorithm approach was used combining a large number of Rhodococcus strains from different families
As no genomic sequence data was available, it was decided to sequence the whole genome to subsequently incorporate this interesting strain into the generated Orthologous Matrix algorithm to further investigate whether this strains produces an Oleate hydratase (Ohy)

Summary

Introduction

Rhodococcus is a genus of aerobic, gram-positive bacteria and is known for its diverse biocatalytic activity towards a plethora of substrates (van der Geize and Dijkhuizen 2004; Jones and Goodfellow 2012; Kim et al 2018; Busch et al 2019). Aromatic or heterocyclic compounds as well as alicyclic hydrocarbons, cholesterol, nitriles and lignin have been shown to be converted by members of the versatile Rhodococcus family (van der Geize and Dijkhuizen 2004; Kim et al 2018). The reason for their catabolic adaptability is explained by interacting factors such as their large genome. The immense progress made in genomic studies enables a fast and extensive processing of bacterial genome information to designate gene functions to undescribed enzymes (Zampolli et al 2019) This helps identifying novel biocatalysts in e.g. Rhodococcus and increases the biotechnological potential for this catalytic powerhouse. They catalyse the reversible water addition to π-bond systems and can be categorised in two groups based on their substrate scope: isolated double bonds or conjugated systems (Resch and Hanefeld 2015; Engleder and Pichler 2018)

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