Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR—Gpr1 was chosen as a prototype to evolve novel glucose sensors. We demonstrate superior variant representation and several unique hits in the synthetic library compared to the PCR generated library. Application of this mutational approach further builds the possibilities of synthetic biology in tuning of a response to known ligands and in generating biosensors for novel ligands.

中文翻译：

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大规模的合成位点饱和GPCR库揭示了改变葡萄糖信号的新型突变。

位点饱和诱变（SSM）是用于蛋白质工程和定向进化实验的强大诱变策略。但是，使用此方法的限制因素是变异的偏倚表示或限制文库大小。为了克服这些障碍，我们使用大规模平行的寡核苷酸合成方法生成了大规模的靶向合成SSM文库，并针对易错（epPCR）文库进行了基准测试。选择酵母葡萄糖激活的GPCR-Gpr1作为开发新型葡萄糖传感器的原型。与PCR生成的文库相比，我们在合成文库中展示了出色的变异体表现形式和几个独特的命中结果。该突变方法的应用进一步建立了合成生物学在调节对已知配体的反应和产生用于新型配体的生物传感器中的可能性。