Abstract
Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.
Highlights
IntroductionChiral alcohols are valuable building blocks for pharmaceuticals and agrochemicals [1] and a multitude of studies have been devoted on biocatalytic methodologies for their production
Chiral alcohols are valuable building blocks for pharmaceuticals and agrochemicals [1] and a multitude of studies have been devoted on biocatalytic methodologies for their production.there is still a high demand for new enzymes, which operate on specific substrates with high activity and selectivity
Vector [15] – that adds an N-terminal His-tag and a TEV protease cleavage site to the protein of interest – with the T7 promoter resulted in approximately identical expression level compared to untagged YlADH2 expressed from pMS470, a vector with tac promoter [16]
Summary
Chiral alcohols are valuable building blocks for pharmaceuticals and agrochemicals [1] and a multitude of studies have been devoted on biocatalytic methodologies for their production. On new alcohol oxidation activities showed, for example, that Yarrowia lipolytica alcohol dehydrogenases (ADHs) are highly interesting candidates for biocatalysis [4]. They are currently filed as putative enzymes [7] Three of these five proteins were annotated as putative ADH1, ADH2, and ADH3, one as a protein with similarity to putative Yarrowia lipolytica ADH3, and one as protein with similarity to mitochondrial ADH3 of S. cerevisiae. Of these proteins, ADH2 showed the highest similarity to ScADH1 and was chosen as a target enzyme (Table 1).
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