Functional characterisation of an essential neo-chromosome III in Sc2.0 strain reveals opportunities and challenges for genome minimisation in Sc3.0

Large-scale genome minimisation in eukaryotes remains a major challenge due to essential genes embedded within deletion-refractory regions and pervasive synthetic lethal interactions. Here, we address these limitations by engineering an essential neo–chromosome III that relocates all 14 essential genes from synthetic chromosome III onto a separate chromosome, thereby enabling further minimisation of synIII. To further expand design space, we created highly synthetic neo-chromosome variants with sequences absent from natural genomes. We refactored essential gene expression using both native and orthogonal promoter–terminator pairs from Saccharomyces paradoxus and S. eubayanus . Reporter assays showed that orthogonal regulatory elements largely recapitulate S. cerevisiae activity. Both architectures restored viability in essential gene deletion libraries, demonstrating robust cross-species complementation. Engineered linear and circular forms of essential neo-chromosomes were highly stable over 100 generations and supported a near wild-type phenotype. Relocating essential functions enabled SCRaMbLE-mediated deletion of previously inaccessible regions, substantially expanding the deletion landscape. To improve the screening efficiency of SCRaMbLEd strains, we developed a SCRaMbLE reporter, ERICA (Elementary Random Integration Cassette), a loxPsym-flanked URA3 cassette that integrates randomly and enables iterative selection. Nanopore sequencing confirmed complex rearrangements, including deletions of up to ∼40 kb and loss of essential loci. Together, this work establishes a modular and extensible platform for orthogonal essential gene engineering and SCRaMbLE-enabled genome reduction, providing key design principles for next-generation synthetic eukaryotic genomes. These findings have broad implications beyond yeast, providing transferable design principles for genome minimisation in more complex eukaryotic systems, including mammalian and human cells.