Widely applicable background depletion step enables transaminase evolution through solid-phase screening.
Matteo PlanchestainerEimear HegartyChristian M HeckmannLouise Jane GourlayFrancesca ParadisiPublished in: Chemical science (2019)
Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.
Keyphrases
- wild type
- crystal structure
- high throughput
- high resolution
- copy number
- induced apoptosis
- crispr cas
- cell cycle arrest
- heavy metals
- wastewater treatment
- molecular docking
- dna methylation
- oxidative stress
- drug induced
- endoplasmic reticulum stress
- molecularly imprinted
- cell proliferation
- solid phase extraction
- risk assessment
- tandem mass spectrometry
- high speed
- sensitive detection
- bioinformatics analysis
- loop mediated isothermal amplification