Genome mining and characterisation of a novel transaminase with remote stereoselectivity

Abstract

Microbial enzymes from pristine niches can potentially deliver disruptive opportunities in synthetic routes to Active Pharmaceutical Ingredients and intermediates in the Pharmaceutical Industry. Advances in green chemistry technologies and the importance of stereochemical control, further underscores the application of enzyme-based solutions in chemical synthesis. The rich tapestry of microbial diversity in the oceanic ecosystem encodes a capacity for novel biotransformations arising from the chemical complexity of this largely unexplored bioactive reservoir. Here we report a novel ω-transaminase discovered in a marine sponge Pseudovibrio sp. isolate. Remote stereoselection using a transaminase has been demonstrated for the first time using this novel protein. Application to the resolution of an intermediate in the synthesis of sertraline highlights the synthetic potential of this novel biocatalyst discovered through genomic mining. Integrated chemico-genomics revealed a unique substrate profile, while molecular modelling provided structural insights into this ‘first in class’ selectivity at a remote chiral centre.

Construction and optimization of a biocatalytic route for the synthesis of neomenthylamine from menthone

“…Leveraging this characteristic, we devised a substrate analog screening strategy, employing the amination of cyclohexanone or its analogs as a model reaction to discover enzymes that can convert (−)-menthone to (+)-neomenthylamine. Subsequently, we screened 83 target enzymes from different sources capable of catalyzing amination reaction, including transaminases (TAs), imine reductases (IREDs), reductive amine enzymes (RedAms), amine dehydrogenases (AmDHs), and aminodeoxychorismate lyases (ADCLs) (Koszelewski et al 2011 ; Tufvesson et al 2012 ; Skalden et al 2015 ; Wetzl et al 2016 ; Guo and Berglund 2017 ; Ramsden et al 2019 ; Gavin et al 2019 ; Mangas-Sanchez et al 2020 ). After expressing and rescreening of the enzymes in the form of cell-free extract, we obtained a transaminase ( Vf TA) from Vibrio fluvialis JS17 (PDB: 5ZTX) that can convert (−)-menthone into (+)-neomenthylamine successfully.…”

Enzyme entrapment, biocatalyst immobilization without covalent attachment

“…Technological advances, in particular genome mining (metagenomics), next generation gene sequencing and bioinformatics, assist the isolation of efficient natural enzyme catalysts. 50,51 Introduction of directed evolution has greatly improved biocatalyst stability and activity. [52][53][54] Computational aided, de novo design has further enhanced the understanding of biocatalysts leading to wider substrate scopes.…”

The Impact of Metagenomics on Biocatalysis

“…Upon examination of the active-site it was found that the transaminase from Pseudovibrio WM33 naturally possessed amino acid substitutions that were introduced into other transaminases to improve reaction rate and melting temperature, showing that metagenomic enzymes can often circumvent tedious engineering efforts. [57] Asymmetric reduction of carbonyls is an essential transformation in biocatalysis. Ketoreductases (KREDs) or alcohol dehydrogenases (ADHs) are able to the reduction of ketones in a wide range of different molecules.…”