Saccharomyces cerevisiae has been demonstrated to be an excellent platform for the multi-fragment assembly of large DNA constructs through its powerful homologous recombination ability. These assemblies have invariably used the stable centromeric single copy vectors. However, many applications of these assembled genomes would benefit from assembly in a higher copy number vector for improved downstream extraction of intact genomes from the yeast. A review of the literature revealed that large multi-fragment assemblies did not appear to have been attempted in multicopy vectors. Therefore, we devised a toolkit that would enable one to seamlessly transition with the same assembling fragments between a single copy and a multicopy vector. We evaluated the assembly of a 28 kb attenuated SARS-CoV-2 genome (lacking the N gene) from 10 fragments in both single copy and multicopy vector systems. Our results reveal that assembly was comparably efficient in the two vector systems. The findings should add to the synthetic biology toolkit of S. cerevisiae and should enable researchers to utilize any of these vector systems depending on their downstream applications.

酿酒酵母通过其强大的同源重组能力已被证明是大型 DNA 构建体多片段组装的绝佳平台。这些组装体总是使用稳定的着丝粒单拷贝载体。然而，这些组装基因组的许多应用将受益于在更高拷贝数载体中的组装，以改善从酵母中提取完整基因组的下游过程。对文献的回顾表明，似乎没有在多拷贝载体中尝试过大型多片段组装。因此，我们设计了一个工具包，使人们能够使用相同的组装片段在单拷贝和多拷贝向量之间无缝转换。我们评估了单拷贝和多拷贝载体系统中 10 个片段组装的 28 kb 减毒 SARS-CoV-2 基因组（缺少 N 基因）。我们的结果表明，两个载体系统中的组装效率相当。这些发现应该添加到酿酒酵母的合成生物学工具包中，并使研究人员能够根据其下游应用来利用任何这些载体系统。