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