Efficient DNA delivery into non-conventional yeasts is a limiting factor for their genetic domestication and use as microbial cell factories. BRIDGE (Bacteria-to-yeast Rapid Interkingdom DNA Gene Exchange) is presented as an integrated synthetic biology platform to overcome this barrier by enabling broad-host interkingdom DNA transfer from bacteria to phylogenetically diverse yeasts. The platform aims to provide a single framework for delivering multigene constructs and accelerating the engineering of previously intractable yeast species.
BRIDGE centers on a compact 6-kb Pan/ARS-oriT broad-host BRIDGE vector that is compatible with the platform workflow. Interkingdom transfer is enabled by a superconjugative helper plasmid, pSC5, which provides the conjugation functions required for bacteria-to-yeast DNA movement. The abstract reports that pSC5 alone enabled access to four genera, while the integrated BRIDGE system expanded that capacity substantially.
Using the BRIDGE system, the authors expanded interkingdom DNA transfer to ten phylogenetically diverse yeast genera. These genera included Saccharomyces, Maudiozyma, Starmerella, Kazachstania, Yarrowia, Zygosaccharomyces, Lachancea, Kluyveromyces, Pichia and Komagataella. The platform therefore demonstrates broad-host delivery across both closely and distantly related yeasts, reflecting its potential utility for genetic domestication across multiple industrially relevant lineages.
BRIDGE establishes an integrated workflow described by the authors as Design-Build-Recover-Deliver. This workflow combines several modular tools: in vitro construction of transcriptional units, single-step in vivo assembly of multigene pathways, rapid plasmid recovery, and broad-host delivery. The reported workflow links established cloning approaches and recovery platforms to enable end-to-end transfer of complex constructs into diverse yeast hosts.
For multigene assembly, individual transcriptional units were constructed using the YeastFab modular cloning system. Complete pathways were assembled by single-step in vivo homologous recombination in Saccharomyces cerevisiae. After assembly, plasmids were rapidly recovered using the EASY-C platform, allowing the constructed multigene vector to be prepared for delivery to recipient yeast genera via BRIDGE.
To demonstrate transfer of a functional multigene pathway, the authors engineered a 16-kb visual reporter comprising a synthetic five-gene violacein biosynthetic pathway derived from Chromobacterium violaceum. This reporter construct was successfully delivered to all ten targeted yeast genera using BRIDGE, showing that full-length multigene synthetic pathways can be moved interkingdom with the platform.
Observed outputs varied by genus. Robust violacein production, which provides a visible purple pigment, was reported across six Saccharomyces species tested and served as a rapid visual marker for transformant identification. Modest functional pathway expression was detected in Kazachstania and Kluyveromyces. In the remaining genera the pathway was transferred successfully but did not produce visible pigmentation, indicating transfer alone does not guarantee detectable pathway output.
The authors highlight that regulatory compatibility between heterologous constructs and host transcriptional/translation systems appears to be the principal determinant of heterologous pathway expression in distantly related yeasts. In other words, while BRIDGE can enable delivery of large, multigene constructs, differences in promoter recognition, regulatory elements, metabolic context, or other host factors limit functional expression in some genera. This distinction underscores that DNA delivery and expression are separable challenges in cross-kingdom engineering.
BRIDGE provides a practical framework to accelerate the genetic modification of yeasts that have been difficult to engineer. By enabling transfer of complex pathways and combining modular cloning, in vivo assembly and rapid plasmid recovery, the platform expands the synthetic biology toolbox available for sustainable biomanufacturing and industrial biotechnology. The capacity to transfer full multigene constructs across diverse hosts could shorten development cycles for new microbial production strains and broaden the set of organisms available for process development.
Details such as quantitative transfer efficiencies, strain-specific parameters, or optimization steps were not reported in the abstract and would require consulting the full preprint or supplementary material. The authors note that transferred pathways may remain nonfunctional in some genera due to regulatory incompatibility rather than failure of DNA transfer. The work is presented as a preprint and has not been certified by peer review.
Collectively, BRIDGE is described as an integrated, modular platform that combines a compact broad-host vector, a superconjugative helper plasmid, modular assembly methods and plasmid recovery to enable rapid interkingdom movement of multigene synthetic pathways into phylogenetically diverse yeasts. The approach prioritizes a complete Design-Build-Recover-Deliver workflow to support genetic domestication efforts and expand options for industrial biotechnology.