Pisera Alexander Olek, Yu Yutong, Williams Rory L, Liu Chang C
Department of Biomedical Engineering, University of California, Irvine, California 92617, United States.
Center for Synthetic Biology, University of California, Irvine, California 92617, United States.
ACS Synth Biol. 2025 Apr 18;14(4):1002-1008. doi: 10.1021/acssynbio.4c00786. Epub 2025 Mar 24.
Efficient methods for diversifying genes of interest (GOIs) are essential in protein engineering. For example, OrthoRep, a yeast-based orthogonal DNA replication system that achieves the rapid diversification of GOIs encoded on a cytosolic plasmid (p1), has been successfully used to drive numerous protein engineering campaigns. However, OrthoRep-based GOI evolution has almost always started from single GOI sequences, limiting the number of locations on a fitness landscape from where evolutionary search begins. Here, we present a simple approach for the high-efficiency integration of GOI libraries onto OrthoRep. By leveraging integrases, we demonstrate recombination of donor DNA onto the cytosolic p1 plasmid at exceptionally high transformation efficiencies, even surpassing the transformation efficiency of standard circular plasmids and linearized plasmid fragments into yeast. We demonstrate our method's utility through the straightforward construction of mock nanobody libraries encoded on OrthoRep, from which rare binders were reliably enriched. Overall, integrase-assisted manipulation of yeast cytosolic plasmids should enhance the versatility of OrthoRep in continuous evolution experiments and support the routine construction of large GOI libraries in yeast, in general.
在蛋白质工程中，使目标基因（GOI）多样化的高效方法至关重要。例如，OrthoRep是一种基于酵母的正交DNA复制系统，可实现胞质质粒（p1）上编码的GOI的快速多样化，已成功用于推动众多蛋白质工程活动。然而，基于OrthoRep的GOI进化几乎总是从单个GOI序列开始，限制了进化搜索起始的适应性景观上的位置数量。在此，我们提出了一种将GOI文库高效整合到OrthoRep上的简单方法。通过利用整合酶，我们证明供体DNA以极高的转化效率重组到胞质p1质粒上，甚至超过标准环状质粒和线性化质粒片段转化到酵母中的效率。我们通过直接构建在OrthoRep上编码的模拟纳米抗体文库证明了我们方法的实用性，从中可靠地富集了稀有结合剂。总体而言，整合酶辅助的酵母胞质质粒操作应增强OrthoRep在连续进化实验中的通用性，并一般支持在酵母中常规构建大型GOI文库。
