Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.
Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.
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DOI：
10.1016/j.jmb.2015.01.003
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发表时间：
2015-03-27
影响因子：
5.6
通讯作者：
DeLisa MP
中科院分区：
文献类型：
--
作者：
Boock JT;King BC;Taw MN;Conrado RJ;Siu KH;Stark JC;Walker LP;Gibson DM;DeLisa MP
许多蛋白质在细菌、酵母和植物中的异源表达往往受到低滴度功能蛋白质的限制。为了解决这个问题，我们在大肠杆菌中创建了一种两级定向进化策略，该策略能够在保持高生物活性的同时优化蛋白质生产。第一层涉及对细胞内蛋白质稳定性的遗传选择，这是基于双精氨酸转位(TAT)途径固有的折叠质量控制机制，而第二层是对蛋白质功能的半高通量筛选。为了证明这一策略的有效性，从植物病原真菌Fusum graminearum中分离出内切葡聚糖酶Cel5A的变异体，其产量比亲本酶提高了30倍。产量的增加归因于两次氨基酸取代，并通过两级方法在两次迭代后被分离出来。在可溶或不可溶的纤维素底物上，活性没有显著的权衡。重要的是，通过将TAT质量控制机制提供的折叠过滤器与基于功能的筛选相结合，我们以不影响催化活性的方式显示了对蛋白质丰度增加的变体的富集性，为设计改进或新功能提供了高度可溶性的亲本。
Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation (Tat) pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, variants of the endoglucanase Cel5A from the plant pathogenic fungus Fusarium graminearum were isolated whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and was isolated after two iterations through the two-tiered approach. There was no significant trade-off in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the Tat quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.