Cloning, Expression And Characterization Of A Novel Nadph-Dependent Carbonyl Reductase From Pichia Pastoris GS115

Optically active alcohols are key building blocks in the preparation of high-value fine chemicals. A straightforward approach to access these enantiomerically pure alcohols is the reduction of the prochiral ketones. The ketone reduction can be conducted biocatalytically or chemically. Biocatalysis has emerged as a great addition to traditional chemical processes for production of some chiral alcohols. Carbonyl reductases catalyze prochiral ketone to afford either R- or S- enantiomers, in practical use the pharmaceuticals are bulky chemicals, so the carbonyl reductases with the activity of bulky chemicals are more valuable.Pichia pastoris GS115 was the most common yeast host used for expression of recombinant proteins, Its genome has been sequenced, and plausible protein coding genes were annotated as well, this facilitates the discovering of novel carbonyl reductases from this strain. By genomic data mining we found a putative carbonyl reductase in Pichia pastoris GS115 chromosome 3 （locus_tag:PAS_chr3₀449）, PasCR was cloned and over-expressed in Escherichia coli Rosseta2, the expressed enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied. The enantio-preference of PasCR follows Prelog’s rule, it strictly uses NADPH as cofactor, and gel filtration suggesting the native form of PasCR is a dimmer. PasCR exhibited highest activity at 35℃ in phosphate buffer at pH 6.5, it asymmetrically reduces a broad range of prochiral ketones such as aromatic ketones and ketoesters to their corresponding alcohol with high enantiomeric purity ee>99%, and have high conversion. It exhibits activity towards some bulky diaryl ketones, for example the enzyme catalyzed 4-methylbenzophenone to afford S- enantiomers with 85% ee value. Also PasCR asymmetric reduces 2-oxo-2-pheylactaldehyde to （R）-1-phenyl-1,2-ethane diol with a conversion of 99% and 99% ee. The ee value and conversion was determinated by chiral HPLC or GC analysis. The configuration of the product was determined by comparing the retention time with the literature or commercially purchased standards. In conclusion the carbonyl reductase from Pichia pastoris GS115 showed broad substrate spectrum, excellent enantio-selectivity and activity to some bulky substrates. Our study provides a valuable guidance for the further application of this versatile biocatalyst in asymmetric synthesis of chiral alcohol with bulky substituents.