Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature
Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature
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DOI：
10.1007/s002530051537
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发表时间：
1999-09-01
影响因子：
5
通讯作者：
Jönsson, LJ
中科院分区：
文献类型：
--
作者：
Cassland, P;Jönsson, LJ
漆酶可用于木质纤维素水解物的酶促脱毒。通过异源表达漆酶，可以获得一株对酚类抑制剂抗性增强，从而提高发酵木质纤维素水解物能力的酿酒酵母菌株。对木质素降解担子菌Trametes versicolor新的漆酶基因lcc2进行了序列分析，结果表明该基因编码一个由499个氨基酸残基组成的同工酶，其前有一个21个残基的信号肽。通过与Edman降解数据的比较，推测LCC2编码一个与漆酶A对应的同工酶。LCC2的基因产物与之前鉴定的杂色葡萄球菌LCC1基因产物有71%的同源性。对漆酶序列的比对表明，与其他白腐菌的同工酶相比，杂色木霉的同工酶与其他白腐菌的同工酶关系更密切。因此，漆酶的多样性是杂色木霉及其近缘种白腐菌的保守特征。在酿酒酵母中表达杂色曲霉Lcc2基因时，活性酶的产生强烈依赖于温度。在培养3天后，当阳性转化子保持在19℃而不是28℃时，漆酶活性提高了16倍。与表达杂色酵母漆酶基因lcc1的毕赤酵母相似的实验也表明，较低的培养温度有利于表达水平的提高，这表明对酿酒酵母表达系统的观察具有普遍意义。
Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 degrees C instead of 28 degrees C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance.