In vitro ribosome synthesis and evolution through ribosome display
In vitro ribosome synthesis and evolution through ribosome display
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DOI：
10.1038/s41467-020-14705-2
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发表时间：
2020-02-28
影响因子：
16.6
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
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作者：
Hammerling, Michael J.;Fritz, Brian R.;Jewett, Michael C.
用于扩大底物掺入和新功能的核糖体的定向进化是具有挑战性的，因为细胞活力的要求限制了可以进行的突变。在这里，我们解决这一挑战相结合的无细胞合成和组装的功能核糖体与核糖体展示开发一个完全在体外的方法核糖体合成和进化（称为RISE）。我们通过从核糖体RNA（rRNA）变体的相似1.7x10（7）成员文库中选择活性基因型，以及从相似4x10（3）rRNA变体文库中鉴定对抗生素克林霉素具有抗性的突变核糖体，来验证RISE方法。我们进一步证明了耐药基因型中正上位性的普遍性，突出了这种相互作用在选择新功能中的重要性。我们预计，RISE将促进分子翻译的理解，并能够选择具有改变性质的核糖体。核糖体的定向进化具有挑战性，因为细胞活力的要求限制了可以进行的突变。在这里，作者开发了一个体外核糖体合成和进化（RISE）平台来克服这些限制。
Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a similar to 1.7x10(7) member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of similar to 4x10(3) rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties. Directed evolution of the ribosome is challenging because the requirement of cell viability limits the mutations that can be made. Here the authors develop a platform for in vitro ribosome synthesis and evolution (RISE) to overcome these constraints.