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Abstract Lignin is a universal waste product of the agricultural industry and is currently seen as a potential feedstock for more sustainable manufacturing. While it is the second most abundant biopolymer in the world, most of it is currently burned as it is a very recalcitrant material. Many recent studies, however, have demonstrated the viability of biocatalysis to improve the value of this feedstock and convert it into more useful chemicals, such as polyhydroxybutyrate, and clean fuels like hydrogen and n-butanol.Rhodopseudomonas palustrisis a gram-negative bacterium which demonstrates a plethora of desirable metabolic capabilities, including aromatic catabolism useful for lignin degradation. This study uses a multi-omics approach, including the first usage of CRISPRi inR. palustris, to investigate the lignin consumption mechanisms ofR. palustris, the essentiality of redox homeostasis to lignin consumption, elucidate a potential lignin catabolic superpathway, and enable more economically viable sustainable lignin valorization processes. 

Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways has proven to be exceedingly difficult, as the cofactors, cellular conditions, and energy sources necessary for this pathway to function may not be present in the host organism. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris , including origins of replication, fluorescent reporters, terminators, and 5′ untranslated regions, and employed the microbe’s endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris ’ many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.