Expression and subcellular localization of (R)- and (S)- specific carbonyl reductases from Candida parapsilosis in Saccharomyces cerevisia

Abstract:

Abstract：[Objective? The (R)- and (S)- specific carbonyl reductases (RCR and SCR) with enhanced green fluorescence protein (EGFP) were expressed in Saccharomyces cerevisiae W303-1A. By analysis of EGFP expression spectrum, the protein distribution and subcellular localization of two enzymes were determined. [Methods? By SOE-PCR method, the fused genes of RCR and SCR with EGFP were cloned and constructed on an eukaryotic expression vector pYX212, and transformed into S. cerevisiae by electroporation. With fluorescent protein as selection marker, the expression and distribution of RCR and SCR were observed. [Results? The EGFP expression in S. cerevisiae cells was observed under the laser scanning confocal microscopy．It was showed that the two enzymes expressed stably in S. cerevisiae and mainly distributed in the intracellular membrane and cytoplasm, while minority were dotted in the center of cells. According to the fluorescent intensity, the expression level of SCR was much higher than that of RCR in S. cerevisiae. The biotransformation results showed that the fused proteins EGFP-RCR and EGFP-SCR reduced 2-hydroxyacetophenone to give (R)- and (S)-1-phenyl-1,2-ethanediol (PED) respectively, with the optical purity of 86.6% in a yield of 70.4% for the former enzyme and with the optical purity of 92.3% in a yield of 81.8% for the latter one. [Discussion? The fusion of RCR or SCR with EGFP showed no effect in protein conformation and biological activity. When compared with the recombinant Escherichia coli harboring RCR or SCR, the genetically engineered S. cerevisiae had obvious advantages in the biological function. The study provided the solid foundations for visible research of functional expression controlling and subcellular localization of carbonyl reductases.