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Abstract 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.
Swidah et al. (Wed,) studied this question.