(70c) Unlocking the Genetic Potential of Rhodopseudomonas Palustris CGA009 for Lignin Valorization

Non-model microorganisms have unique and robust biochemical processes that are unavailable in their model counterparts. Harnessing their unique capabilities have the potential to enable the sustainable synthesis of numerous products; yet, the ability to precisely and reliably engineer these microbes is hampered by their innate repair and immune systems as well as a lack of synthetic biology tools. The purple non-sulfur bacterium Rhodopseudomonas palustris CGA009 employs four different metabolic modes, photoautotrophic, photoheterotrophic, chemoautotrophic, and chemoheterotrophic, to adapt to a diversity of environmental conditions. In addition to fixing carbon dioxide during autotrophic growth, the bacterium can also degrade recalcitrant aromatic compounds derived from lignin, using both aerobic and anaerobic pathways. R. palustris is the only bacterium that encodes two carbon dioxide-fixing RubisCO-like proteins and it is one of two known bacteria that encodes all three nitrogen-fixing nitrogenase isozymes. Purple non-sulfur bacteria perform anoxygenic photosynthesis which allows oxygen-sensitive nitrogen fixation to occur at the same time. Furthermore, even in very low intensity light R. palustris can remain metabolically active in a non-growing state for months. Published engineering efforts that unlock R. palustris’ genetic potential to valorize lignin breakdown products have been minimal, possibly due to the bacterium’s intrinsic antibiotic resistance, the genetic instability of exogenous DNA, and the lack of precise orthogonal regulators of gene expression. Multiple tracks are being pursued to address these limitations. In addition to characterizing the microbe’s antibiotic resistance, genes for antibiotic-modifying enzymes have been removed from the genome. Methylation patterns of heterologous plasmids are being explored, already with a dramatic improvement in transformation efficiency. Neutral sites for genomic integration of synthetic devices are being characterized. The impact of genes in the microbe’s mutagenic translation synthesis system on the genetic stability of exogenous DNA are also being investigated through knockout strains. Finally, synthetic biology tools for transcriptional regulation are being developed to explore R. palustris’ catabolic pathways for lignin-derived compounds and bioplastic production. This work will contribute to the development of this promising bacterium as a biotechnology platform and to the generation of important design guidelines for engineering non-model microorganisms.