Biology subjects

Swidah, R.

Publications and source records attributed to Swidah, R..

4 recordsLinked to original sources

bioRxiv2026

BRIDGE: A broad-host platform for interkingdom DNA delivery enabling the genetic domestication of phylogenetically diverse yeasts

Efficient DNA delivery remains one of the greatest barriers to engineering non-conventional yeasts, limiting their genetic domestication and exploitation as next-generation microbial cell factories. Here, we present BRIDGE (Bacteria-to-yeast Rapid Interkingdom DNA Gene Exchange), an integrated synthetic biology platform for broad-host interkingdom DNA delivery. BRIDGE is centred on a compact 6-kb Pan/ARS-oriT broad-host BRIDGE vector, enabled by a superconjugative helper plasmid (pSC5). Using BRIDGE, we expanded interkingdom DNA transfer from representatives of four genera accessible using pSC5 alone to ten phylogenetically diverse yeast genera, including Saccharomyces, Maudiozyma, Starmerella, Kazachstania, Yarrowia, Zygosaccharomyces, Lachancea, Kluyveromyces, Pichia and Komagataella. To demonstrate the versatility of BRIDGE, we engineered a 16-kb visual reporter carrying a synthetic five-gene violacein biosynthetic pathway from Chromobacterium violaceum. Individual transcriptional units were first constructed using the YeastFab modular cloning system and subsequently assembled into the complete pathway by single-step in vivo homologous recombination in Saccharomyces cerevisiae, followed by rapid plasmid recovery using the EASY-C platform. The reporter was successfully delivered to all ten yeast genera, demonstrating BRIDGE-mediated transfer of complete multigene synthetic pathways. Robust violacein production was observed across all six Saccharomyces species tested, providing a rapid visual marker for transformant identification, while modest functional pathway expression was also detected in Kazachstania and Kluyveromyces. Successful pathway transfer, albeit without visible pigmentation, was achieved in the remaining genera, indicating that regulatory compatibility, rather than DNA transfer, is the principal determinant of heterologous pathway expression in distantly related yeasts. Collectively, BRIDGE establishes an integrated Design-Build-Recover-Deliver workflow that combines modular in vitro construction of transcriptional units, single-step in vivo assembly of multigene pathways, rapid plasmid recovery using the EASY-C platform and broad-host interkingdom DNA delivery. This versatile framework enables the rapid genetic modification of previously intractable microorganisms, expanding the synthetic biology toolbox for sustainable biomanufacturing and industrial biotechnology.

synthetic biology↗

bioRxiv2026

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

AbstractLarge-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.

genomics↗

bioRxiv2022

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the [~]190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

synthetic biology↗

bioRxiv2022

Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE system to the newly synthesized Sc2.0 megabase chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by i) removing most of the chromosome content, or ii) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic yeast resource opens new paradigms in studying aneuploidy. In briefUse of SCRaMbLE and newly synthesized Mb-scale Sc2.0 chromosome VII enables insights into genotype/phenotype relationships associated with aneuploidy HighlightsO_LIDe novo design and synthesis of a Mb-scale synthetic yeast chromosome VII, carrying 11.8% sequence modifications and representing nearly 10% of the yeast genome. C_LIO_LIA disomic yeast (n + synVII) is constructed for dissecting the aneuploidy phenotype C_LIO_LISCRaMbLE enables systematic exploration of regions causing aneuploidy phenotypes C_LIO_LIChromosomal copy number and content both contribute to aneuploidy phenotypes C_LIO_LIA 20 Kb deletion on the right arm of synVII leads to fitness improvement linked to up-regulation of protein synthesis C_LI

synthetic biology↗