Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Appl Biochem Biotechnol. 2010 Mar;160(3):868-78. doi: 10.1007/s12010-009-8528-9. Epub 2009 Feb 10.
An R-specific carbonyl reductase from Candida parapsilosis (CprCR) catalyzes the transformation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone. The gene rcr coding CprCR contains a few codons rarely used by Escherichia coli. In order to improve chiral alcohol production, three codon variants Delta24, aRCR, and mRCR of CprCR were designed through truncation of 4-27 bp disorder sequence at the 5'-terminus or/and adaption of nine rare codons. The effects of codon optimization on enzyme activity, protein production, and biotransformation were studied. Among these three types, the disorder sequence-truncated and rare codon-adapted variant mRCR presents the highest enzyme activity. When compared with CprCR, mRCR showed an increase of 35.6% in the total activity of cell-free extracts. The specific activity of mRCR presented similar increase in the cell-free extract with purified protein, which suggested that the codon optimization caused positive effect on protein productivity of variant enzyme. When microbial cells concentration was 30% (w/v), the molar conversion yield and enantiomeric excess of the mRCR variant reached 86.4% and 93.6%, which were increased 36.5% and 15.8% than those of wild-type at a high substrate concentration of 5 g/L. The work will supply a new method for improving chiral alcohol preparation with codon engineered microorganisms.
来自近平滑假丝酵母（Candida parapsilosis）的一种特定于 R 的羰基还原酶（CprCR）催化（R）-1-苯基-1,2-乙二醇从 2-羟基苯乙酮的转化。编码 CprCR 的 rcr 基因包含一些大肠杆菌很少使用的密码子。为了提高手性醇的产量，通过在 5'端截断 4-27bp 的无序序列和/或适应九个稀有密码子，设计了 CprCR 的三个密码子变体 Delta24、aRCR 和 mRCR。研究了密码子优化对酶活性、蛋白质产量和生物转化的影响。在这三种类型中，无序序列截断和稀有密码子适应的变体 mRCR 表现出最高的酶活性。与 CprCR 相比，mRCR 在无细胞提取物的总活性中增加了 35.6%。mRCR 在无细胞提取物中的比活性也呈现出相似的增加，这表明密码子优化对变体酶的蛋白质产率产生了积极的影响。当微生物细胞浓度为 30%（w/v）时，mRCR 变体的摩尔转化率和对映体过量值分别达到 86.4%和 93.6%，在 5g/L 的高底物浓度下，分别比野生型提高了 36.5%和 15.8%。这项工作将为利用基因工程微生物制备手性醇提供一种新方法。
