Attenuated secretion of the thermostable xylanase xynB from Pichia pastoris using synthesized sequences optimized from the preferred codon usage in yeast.
Attenuated secretion of the thermostable xylanase xynB from Pichia pastoris using synthesized sequences optimized from the preferred codon usage in yeast.
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DOI：
10.4014/jmb.1109.09032
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发表时间：
2012-03
影响因子：
2.8
通讯作者：
Yuankai Huang;Yaosheng Chen;Delin Mo;P. Cong;Zuyong He
中科院分区：
文献类型：
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作者：
Yuankai Huang;Yaosheng Chen;Delin Mo;P. Cong;Zuyong He
木聚糖酶在工农业领域有着广泛的应用。然而，本地品种的低产量木聚糖酶不能满足日益增长的市场需求。因此，通过基础基因优化提高木聚糖酶的外源表达可能有助于克服这一不足。在本研究中，我们从橄榄绿链霉菌A1中合成了高gc含量的耐热木聚糖酶基因xynB的天然序列，并设计了一个稍微偏向at的序列，其密码子完全优化为有利于毕赤酵母。通过检测单拷贝数整合物来比较这些序列在P. pastoris X33中的表达效率，并使用qPCR对其进行定量。令人惊讶的是，高GC含量似乎并不不利于xynB在酵母中的异源表达，而优化后的序列，其密码子使用极度偏斜，在酵母细胞中表现出更丰富的合成重组蛋白积累，但从酶活性分析推断，其分泌水平降低了约30%。在这项研究中，我们开发了一种更准确的方法来比较单个酵母转化子的表达水平。此外，我们的结果为进一步研究异种表达和分泌蛋白的合理设计策略提供了一个实际的例子。
Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.