(450a) Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas Palustris

Model organisms have been used as a biological, more sustainable alternative for manufacturing products such as pharmaceuticals, cosmetics, surfactants, and fragrances. However, these organisms are often metabolically restrained to only catabolizing simple sugars, and bioprocesses involving model organisms are often required to have low stress conditions. Non-model organisms possess many unique biochemical capabilities. Some of these organisms are capable of photosynthesis, can fix carbon dioxide and nitrogen, produce hydrogen and bioplastics, and can catabolize recalcitrant feedstocks such as lignin monomers and methane. Efforts to transfer metabolic pathways from non-model organisms to model organisms however have been fraught with difficulties. The cofactors, cellular conditions, as well as the energy and carbon sources needed for heterologous pathways to function may not be present in the host model bacterium. As a result, these pathways do not perform as well as in other microorganisms. The metabolically versatile non-model organism Rhodopseudomonas palustris is a purple non-sulfur soil bacterium which can produce hydrogen, is capable of all four modes of metabolism, and can catabolize lignin derived aromatic compounds. R. palustris is also capable of carbon dioxide fixation, as it possesses two forms of RuBisCo, and utilizes three nitrogenase isozymes to fix nitrogen while simultaneously producing hydrogen. This chassis benefits from an extreme metabolic versatility, yet stable heterologous gene expression from this bacterium has been problematic due to its high intrinsic resistance to several antibiotics. There is also a lack of synthetic biology parts that have been investigated in this microbe. We have provided a synthetic biology toolbox for R. palustris by including several synthetic biology parts such as origins of replication, terminators, and 5’ untranslated regions, characterizing the fluorescence strengths of various fluorescent reporters, as well as the utilization of R. palustris’ endogenous plasmid for heterologous protein production. This work determines the efficacy of a variety of synthetic biology tools for engineering R. palustris and harnessing its many unique biochemical processes. This study has also helped define the principles by which this microorganism can be engineered to express heterologous genes through a methodology that could be applied to other non-model microorganisms. This work has been published in the Journal of Frontiers in Bioengineering and Biotechnology, and can be found at https://doi.org/10.3389/fbioe.2022.800734.