(R)-、(S)-羰基还原酶在酿酒酵母中的表达和亚细胞定位

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

摘要:

摘要：【目的】将增强型荧光蛋白标记的(R)-和(S)-羰基还原酶于酿酒酵母（Saccharomyces cerevisiae W303-1A）细胞中表达，分析荧光蛋白表达谱，确定两种酶在细胞中的功能分布和亚细胞定位。【方法】采用SOE-PCR法克隆出增强型荧光蛋白与(R)-和(S)-羰基还原酶的融合基因，构建到真核表达载体pYX212中，电击转化酵母细胞，以荧光蛋白为筛选标志，观察两种酶在酵母细胞中的表达和分布。【结果】激光扫描共聚焦显微观察表明(R)-和(S)-羰基还原酶多定位于细胞内膜和细胞质中稳定表达，少数成点状分布于细胞中央。根据荧光强度可知(S)-羰基还原酶的表达水平明显高于(R)-羰基还原酶。生物转化结果显示融合型(R)-和(S)-羰基还原酶催化底物2-羟基苯乙酮，分别获得(R)-和(S)-苯基乙二醇，前者产物的光学纯度和产率为86.6%和70.4%，后者产物的光学纯度和产率分别为92.3%和81.8%。【讨论】荧光蛋白与酶的融合没有改变靶蛋白的分子构象与生物活性，酿酒酵母工程菌较重组大肠杆菌具有更明显的生物功能优势，该研究为羰基还原酶蛋白的功能表达调控与亚细胞定位的可视化研究奠定了坚实的基础。

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.