Thayer School of Engineering, Dartmouth College, Hanover, USA.
BMC Biotechnol. 2009 Nov 20;9:95. doi: 10.1186/1472-6750-9-95.
In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing.
It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion.
These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.
除了提供转录和翻译的分子机制外，重组微生物表达宿主还保持着至关重要的基因型-表型联系，这对于高通量筛选和回收质粒文库编码的蛋白质至关重要。已知大肠杆菌细胞可以同时转化多个独特的质粒，从而使重组文库筛选实验变得复杂。由于它们可能产生误导结果，因此在以前的模型研究中已经对细菌多载体转化体进行了彻底的特征描述。与细菌系统相比，关于酵母中多载体转化体的定量信息很少。酿酒酵母是用于细胞表面展示、组合蛋白工程和其他重组文库筛选的最广泛使用的真核平台。为了描述这个重要宿主中多载体转化体的程度和性质，通过 DNA 测序分析了通过酵母同源重组构建的质粒源性基因文库。
研究发现，酵母同源重组文库中的克隆多达 90%可能是多载体转化体，这些克隆平均携带四个或更多独特的突变基因，并且在液体生长过程中，这些多载体细胞在 24 小时以上的时间内作为文库群体的重要比例持续存在。载体浓度和载体与插入物的比例均影响文库中多载体转化体的比例，但它们的群体频率与转化效率无关。有趣的是，多载体转化体产生的质粒平均数量与其文库群体比例无关。
这些结果突出了多载体转化体主导同源重组构建的酵母文库的潜力。以前未被认识到的多转化酵母细胞的普遍性和持久性对酵母文库筛选具有重要意义。本文描述的定量信息应提高对该问题的认识，并且为这些研究开发的快速测序方法应该广泛用于识别多载体转化体并避免与获得一个以上独特质粒的细胞相关的并发症。
