Construction of a Yeast Cell-Surface Display System and Expression of Trametes sp. laccase
Construction of a Yeast Cell-Surface Display System and Expression of Trametes sp. laccase
复制标题
酵母细胞表面展示系统的构建和栓菌的表达。
DOI：
10.4028/www.scientific.net/amr.347-353.3635
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
G. He
中科院分区：
文献类型：
--
作者：
J. Lu;Qin Guo;M. Cui;Lu Yang;S. Du;H. Ruan;G. He
漆酶（1.10.3.2，p-diphenol：dioxygen oxidoreductases）是一种常见于细菌、真菌和植物中的含蓝铜氧化酶家族。它能够氧化和降解各种芳香族化合物和其他有机化合物。由于这种能力，漆酶可以用于环境生物修复过程和工业目的。酶的细胞表面展示是酵母中最具吸引力的应用之一。构建全细胞生物催化剂是一种有效的利用方法。根据意大利酿酒酵母（Saccharomyces Italic textcerevisiae）的密码子偏好性，对Trametes sp. C30 LAC 3的cDNA序列进行了优化和合成。扩增编码半乳糖激酶（GAL 1）启动子、α交配因子1（MFα1）前原分泌信号、完全密码子优化的LAC 3、α凝集素C端320个氨基酸、乙醇脱氢酶（ADH 1）终止子和kanMX表达盒的基因，并克隆到YEplac 181中，构建以α凝集素为锚的细胞表面展示载体pGMAAK-lac 3。将pGMAAK-lac 3转化S.啤酒。结果表明，LAC 3在S.啤酒。但底物特异性和活性发生了明显变化。展示的LAC 3失去了对酚类底物（愈创木酚）的活性，并且其对非酚类底物（ABTS）的活性大大降低。据我们所知，这是首次尝试通过细胞表面展示技术构建和表达漆酶。
Laccases (1.10.3.2, p-diphenol: dioxygen oxidoreductases) is a family of blue copper-containing oxidases that are commonly found in bacteria, fungi and plants. It is able to oxidize and degrade a variety of aromatic compounds and other organic compounds. Due to this ability, laccases can serve environmental bioremediation processes and industrial purposes. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a effective utilization to construct the whole cell biocatalyst. The cDNA sequence of Trametes sp. C30 LAC3 was optimized and synthesized according to the codon bias of Saccharomyces Italic textcerevisiae, because codon optimization has been proved to be effective to maximize production of heterologous proteins in yeast. The genes encoding galactokinase (GAL1) promoter, α-mating factor 1 (MFα1) pre-pro secretion signal, fully codon-optimized LAC3, the 320 amino acids of C terminal of α-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-lac3 with α-agglutinin as an anchor. Then pGMAAK-lac3 was transformed into S. cerevisiae. The results show LAC3 was immobilized and actively expressed on S. cerevisiae. However, the substrate specifity and activity were obviously changed. The displayed LAC3 lost the activity to phenolic substrate (guaiacol) and its activity to non-phenolic substrate (ABTS) was greatly reduced. To our knowledge, this was the first attempt to construct and express laccase through cell-surface display technology.