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1. Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model

   https://doi.org/10.1128/spectrum.01463-22  

   Chowdhury, Niaz Bahar ; Alsiyabi, Adil ; Saha, Rajib ( June 2022 , Microbiology Spectrum)

   Gralnick, Jeffrey A. (Ed.)

   ABSTRACT Rhodopseudomonas palustris CGA009 is a Gram-negative purple nonsulfur bacterium that grows phototrophically by fixing carbon dioxide and nitrogen or chemotrophically by fixing or catabolizing a wide array of substrates, including lignin breakdown products for its carbon and fixing nitrogen for its nitrogen requirements. It can grow aerobically or anaerobically and can use light, inorganic, and organic compounds for energy production. Due to its ability to convert different carbon sources into useful products during anaerobic growth, this study reconstructed a metabolic and expression (ME) model of R. palustris to investigate its anaerobic-photoheterotrophic growth. Unlike metabolic (M) models, ME models include transcription and translation reactions along with macromolecules synthesis and couple these reactions with growth rate. This unique feature of the ME model led to nonlinear growth curve predictions, which matched closely with experimental growth rate data. At the theoretical maximum growth rate, the ME model suggested a diminishing rate of carbon fixation and predicted malate dehydrogenase and glycerol-3 phosphate dehydrogenase as alternate electron sinks. Moreover, the ME model also identified ferredoxin as a key regulator in distributing electrons between major redox balancing pathways. Because ME models include the turnover rate for each metabolic reaction, it was used to successfully capture experimentally observed temperature regulation of different nitrogenases. Overall, these unique features of the ME model demonstrated the influence of nitrogenases and rubiscos on R. palustris growth and predicted a key regulator in distributing electrons between major redox balancing pathways, thus establishing a platform for in silico investigation of R. palustris metabolism from a multiomics perspective. IMPORTANCE In this work, we reconstructed the first ME model for a purple nonsulfur bacterium (PNSB). Using the ME model, different aspects of R. palustris metabolism were examined. First, the ME model was used to analyze how reducing power entering the R. palustris cell through organic carbon sources gets partitioned into biomass, carbon dioxide fixation, and nitrogen fixation. Furthermore, the ME model predicted electron flux through ferredoxin as a major bottleneck in distributing electrons to nitrogenase enzymes. Next, the ME model characterized different nitrogenase enzymes and successfully recapitulated experimentally observed temperature regulations of those enzymes. Identifying the bottleneck responsible for transferring an electron to nitrogenase enzymes and recapitulating the temperature regulation of different nitrogenase enzymes can have profound implications in metabolic engineering, such as hydrogen production from R. palustris . Another interesting application of this ME model can be to take advantage of its redox balancing strategy to gain an understanding of the regulatory mechanism of biodegradable plastic production precursors, such as polyhydroxybutyrate (PHB). 

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

   https://doi.org/10.3389/fbioe.2022.800734  

   Immethun, Cheryl M. ; Kathol, Mark ; Changa, Taity ; Saha, Rajib ( March 2022 , Frontiers in Bioengineering and Biotechnology)

   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. 

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3. Kinetic modeling of anaerobic degradation of plant-derived aromatic mixtures by Rhodopseudomonas palustris

   https://doi.org/10.1007/s10532-021-09932-3  

   Ma, Yanjun ; Donohue, Timothy J. ; Noguera, Daniel R. ( April 2021 , Biodegradation)

   null (Ed.)

   Abstract Rhodopseudomonas palustris is a model microorganism for studying the anaerobic metabolism of aromatic compounds. While it is well documented which aromatics can serve as sole organic carbon sources, co-metabolism of other aromatics is poorly understood. This study used kinetic modeling to analyze the simultaneous degradation of aromatic compounds present in corn stover hydrolysates and model the co-metabolism of aromatics not known to support growth of R. palustris as sole organic substrates. The simulation predicted that p -coumaroyl amide and feruloyl amide were hydrolyzed to p -coumaric acid and ferulic acid, respectively, and further transformed via p -coumaroyl-CoA and feruloyl-CoA. The modeling also suggested that metabolism of p -hydroxyphenyl aromatics was slowed by substrate inhibition, whereas the transformation of guaiacyl aromatics was inhibited by their p -hydroxyphenyl counterparts. It also predicted that substrate channeling may occur during degradation of p -coumaroyl-CoA and feruloyl-CoA, resulting in no detectable accumulation of p -hydroxybenzaldehyde and vanillin, during the transformation of these CoA ligated compounds to p- hydroxybenzoic acid and vanillic acid, respectively. While the simulation correctly represented the known transformation of p -hydroxybenzoic acid via the benzoyl-CoA pathway, it also suggested co-metabolism of vanillic acid and syringic acid, which are known not to serve as photoheterotrophic growth substrate for R. palustris . 

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4. Stromal NADH supplied by PHOSPHOGLYCERATE DEHYDROGENASE3 is crucial for photosynthetic performance

   https://doi.org/10.1093/plphys/kiaa117  

   Höhner, Ricarda ; Day, Philip M ; Zimmermann, Sandra E ; Lopez, Laura S ; Krämer, Moritz ; Giavalisco, Patrick ; Correa Galvis, Viviana ; Armbruster, Ute ; Schöttler, Mark Aurel ; Jahns, Peter ; et al ( January 2021 , Plant Physiology)

   Abstract During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin–Benson–Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis. 

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5. Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants

   https://doi.org/10.1128/AEM.00487-21  

   Moxley, W. Chris ; Eiteman, Mark A. ( June 2021 , Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli , pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase ( ldhA ) and pyruvate oxidase ( poxB ). The effect of deleting phosphoenolpyruvate synthase ( ppsA ) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h −1 , an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli . This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes. 

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