Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution
Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution
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DOI：
10.1128/aem.69.2.987-995.2003
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发表时间：
2003-02-01
影响因子：
4.4
通讯作者：
Arnold, FH
中科院分区：
文献类型：
--
作者：
Bulter, T;Alcalde, M;Arnold, FH
来自嗜热毁丝霉 (MtL) 的漆酶在酿酒酵母中以功能形式表达。定向进化将酵母漆酶的表达提高了八倍，达到迄今为止报道的最高水平（18 毫克/升）。加上 k(cat) 增加 22 倍，总活性增强了 170 倍。 MtL突变体对2,2'-连氮基-双(3-乙基苯并噻唑啉-6-磺酸)和丁香醛连氮的特异活性表明底物特异性并未因引入的突变而改变。最有效的突变（总活性增加10倍）在蛋白质的C端加工位点引入了Kex2蛋白酶识别位点，调整蛋白质序列以适应异源宿主的不同蛋白酶特异性。 C 末端被证明对漆酶活性很重要，因为通过截断基因去除它会使活性降低六倍。为了获得更高的活性，在九代进化过程中积累的突变降低了酶的稳定性。在一代中筛选改进的稳定性产生了比异源野生型更稳定并保留改进的活性的突变体。酿酒酵母中表达的 MtL 的分子量比嗜热毁丝酵母中表达的相同酶的分子量高 30%（110 kDa 与 85 kDa）。过度糖基化（对应于一个 N-糖基化位点上的 120 个单体聚糖）是造成这种增加的原因。这种酿酒酵母表达系统使 MtL 可用于通过定向进化进行功能定制。
Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.