Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris

Abstract

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.

Protocol to develop a synthetic biology toolkit for the non-model bacterium R. palustris

“… Note: Figure 3 from Immethun & Kathol et al. 1 depicts an example of a trimmed RBS library, where a linear gradient of expression level is made by selecting various RBS sequences with differing expression levels from an initial scan. Figure 3 A shows the initial collection of colonies that were picked from plates, while Figure 3 B depicts the trimmed RBS library.…”

“…Figure 3 A shows the initial collection of colonies that were picked from plates, while Figure 3 B depicts the trimmed RBS library. 1

Figure 3 RBS library fluorescence scans (A) Total single colonies picked from transformed R. palustris cultures with replaced RBS from Immethun and Kathol et al. 1 Fluorescence of each culture was determined by performing a fluorescence marker expression test and measured using a plate reader.

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“…This protocol may also be applicable for other non-model organisms. For complete details on the use and execution of this protocol, please refer to Immethun et al.. 1 …”

A coarse-grained bacterial cell model for resource-aware analysis and design of synthetic gene circuits

“…While the overwhelming majority of proteins is not actively degraded in the bacterial cell [33], mRNA degradation, conversely, is widespread and happens at a high rate [34]. Hence, we include additional constant degradation terms β a and β r in Equations ( 1)- (2), which describe mRNA concentration dynamics.…”

“…As for any engineered system, predictable behavior is a key requirement for synthetic biology designs. However, the forecasting of their performance is complicated by the interconnectedness of biological processes in living cells [2,3,4]. Indeed, even the gene circuit components that do not engage in direct biological interactions may exhibit interdependence caused by indirect couplings via shared cellular resources.…”