Summary Whether synthetic genomes can 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 report de novo synthesis of the largest eukaryotic chromosome thus far, synIV , a 1,454,621-bp Saccharomyces cerevisiae chromosome resulting from extensive genome streamlining and modification. During the construction of synIV , 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. Graphical abstract Highlights De novo synthesis of the largest eukaryotic chromosome, synIV SynIV shows similar 3D structure to wild-type IV, despite thousands of changes made to it “Inside-out” repositioning of synIV in nucleus shows minor transcriptional changes Multipoint tethering synIV to inner nuclear membrane represses transcription of whole chromosome

Manipulating the 3D organization of the largest synthetic yeast chromosome