Manipulating the 3D organization of the largest synthetic yeast chromosome [RNAseq]

Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.合成基因组能否支撑生命活动，一直是合成生物学领域广受关注的研究课题，尤其当合成基因组被数以千计的定制化特征进行大规模修饰后。本研究报道了迄今为止规模最大的真核染色体synIV的从头合成：该染色体全长1,454,621 bp，为经大规模基因组精简与修饰得到的酿酒酵母（Saccharomyces cerevisiae）染色体。在synIV的构建过程中，我们开发了结合分级整合策略的大片段组装技术，该技术显著提升了合成染色体构建的准确性与灵活性，并简化了染色体调试流程。除通过序列重写对synIV进行大幅改造外，我们还对酿酒酵母细胞核内synIV的三维结构进行人工操控，以探索核空间内的基因调控机制。令人意外的是，我们仅观测到极少量基因表达变化，这表明酵母核质内的染色体定位对基因调控的影响较小。最后，我们通过synIV上数百个loxPsym位点将其锚定至内核膜，观测到整条染色体的转录抑制现象，证明了无需改变DNA序列即可实现全染色体范围的转录操控。本研究对最大合成酵母染色体空间结构的操控，为合成基因组的高阶架构设计提供了重要参考。