The development of Sinorhizobium meliloti and Deinococcus radiodurans as chassis for synthetic biology applications

Microorganisms can be harnessed for bioproduction and biotechnology to further global efforts in agriculture, health, manufacturing and sustainability. Traditional microbial chassis used for these purposes are the most well-characterized bacteria and yeast, Escherichia coli and Saccharomyces cerevisiae. Synthetic biology can be used to facilitate engineering of microbial chassis with new or improved traits. However, there is a need to expand the number and diversity of available microbial chassis to include microorganisms that have innate genetic, metabolic and physiological characteristics that we could make use of. Two such bacteria include the nitrogen-fixing plant symbiont, Sinorhizobium meliloti, and the polyextremophile, Deinococcus radiodurans. While some progress has been made toward the development of these chassis, further strain and tool developments are required to unlock their full potential. Here, I present the expansion of the genetic toolkits for S. meliloti and D. radiodurans to improve their utility as bacterial chassis for synthetic biology applications. First, I engineered a genome-reduced strain of S. meliloti as a novel conjugative host and demonstrated the transfer of multi-host shuttle vectors to bacteria, yeast and microalgae. Then, I developed a conjugative protocol to transfer DNA from E. coli to D. radiodurans and showed its utility through the generation of robust systems for conjugation-based genome engineering and whole genome cloning in vivo. Using this method, I cloned the large (178 kb) MP1 megaplasmid from D. radiodurans in E. coli. Finally, I developed a strategy to create seamless gene deletions in D. radiodurans which was demonstrated through the sequential genetic knockout of four restriction-modification systems. The tools and strains developed in my thesis will add to the growing genetic toolkits of S. meliloti and D. radiodurans. The establishment of S. meliloti as an alternative chassis for interkingdom DNA transfer will allow for the study and engineering of agriculturally-relevant microorganisms and modulation of microbial communities. Likewise, the expansion of genetic tools will establish D. radiodurans as a microbial platform for industrial applications and the study of extremophile biology. These bacterial chassis will complement the use of traditional microbial chassis, broadening the potential solutions synthetic biology could offer

Microbial applications for sustainable space exploration beyond low Earth orbit

Abstract With the construction of the International Space Station, humans have been continuously living and working in space for 22 years. Microbial studies in space and other extreme environments on Earth have shown the ability for bacteria and fungi to adapt and change compared to “normal” conditions. Some of these changes, like biofilm formation, can impact astronaut health and spacecraft integrity in a negative way, while others, such as a propensity for plastic degradation, can promote self-sufficiency and sustainability in space. With the next era of space exploration upon us, which will see crewed missions to the Moon and Mars in the next 10 years, incorporating microbiology research into planning, decision-making, and mission design will be paramount to ensuring success of these long-duration missions. These can include astronaut microbiome studies to protect against infections, immune system dysfunction and bone deterioration, or biological in situ resource utilization (bISRU) studies that incorporate microbes to act as radiation shields, create electricity and establish robust plant habitats for fresh food and recycling of waste. In this review, information will be presented on the beneficial use of microbes in bioregenerative life support systems, their applicability to bISRU, and their capability to be genetically engineered for biotechnological space applications. In addition, we discuss the negative effect microbes and microbial communities may have on long-duration space travel and provide mitigation strategies to reduce their impact. Utilizing the benefits of microbes, while understanding their limitations, will help us explore deeper into space and develop sustainable human habitats on the Moon, Mars and beyond

Cloning of Thalassiosira pseudonana’s Mitochondrial Genome in Saccharomyces cerevisiae and Escherichia coli

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

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer.

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 105 and 2.1 x 103 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

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

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