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

Abstract

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.