Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae . Here, we report the development of Sinorhizobium meliloti , a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E . coli , S . cerevisiae , and the eukaryotic microalgae Phaeodactylum tricornutum . To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S . meliloti that enable DNA transformation with up to 1.4 x 10 5 and 2.1 x 10 3 CFU μg -1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S . meliloti , E . coli , S . cerevisiae , and P . tricornutum . We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S . meliloti to E . coli , S . cerevisiae , and P . tricornutum . The development of S . meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture.

HIGHLIGHTS• TAR-cloned mitochondrial genome of P. tricornutum in yeast • Developed PCR-based cloning method to create designer algal mitochondrial genomes • Stably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts . CC-BY-NC-ND 4.

Fungi are nature’s recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.