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1. Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae

   https://doi.org/10.3389/fmicb.2022.849573  

   Lee, Jessica A.; Stolyar, Sergey; Marx, Christopher J. (March 2022, Frontiers in Microbiology)

   Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C 1 ) compounds. The C 1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium . We demonstrated growth on p -hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium , but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans , and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation. 

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2. Characterizing the interplay of rubisco 1 and nitrogenase enzymes in anaerobic-photoheterotrophically grown Rhodopseudomonas palustris CGA009 through a genome-scale metabolic and expression model

   https://doi.org/10.1101/2022.03.03.482919  

   Chowdhury, Niaz B.; Alsiyabi, A.; Saha, R. (March 2022, bioRxiv)

   Rhodopseudomonas palustris CGA009 (R. palustris) is a gram negative purple non-sulfur bacteria that grows phototrophically or chemotrophically by fixing or catabolizing a wide array of substrates including lignin breakdown products (e.g., p-coumarate) for its carbon and 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 in anaerobic mode, this study, for the first time, 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. Since ME-models include 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 multi-omics perspective. 

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3. Coordinated regulation of the entry and exit steps of aromatic amino acid biosynthesis supports the dual lignin pathway in grasses

   https://doi.org/10.1038/s41467-023-42587-7  

   El-Azaz, Jorge; Moore, Bethany; Takeda-Kimura, Yuri; Yokoyama, Ryo; Wijesingha Ahchige, Micha; Chen, Xuan; Schneider, Matthew; Maeda, Hiroshi A. (November 2023, Nature Communications)

   Abstract Vascular plants direct large amounts of carbon to produce the aromatic amino acid phenylalanine to support the production of lignin and other phenylpropanoids. Uniquely, grasses, which include many major crops, can synthesize lignin and phenylpropanoids from both phenylalanine and tyrosine. However, how grasses regulate aromatic amino acid biosynthesis to feed this dual lignin pathway is unknown. Here we show, by stable-isotope labeling, that grasses produce tyrosine >10-times faster than Arabidopsis without compromising phenylalanine biosynthesis. Detailed in vitro enzyme characterization and combinatorialin plantaexpression uncovered that coordinated expression of specific enzyme isoforms at the entry and exit steps of the aromatic amino acid pathway enables grasses to maintain high production of both tyrosine and phenylalanine, the precursors of the dual lignin pathway. These findings highlight the complex regulation of plant aromatic amino acid biosynthesis and provide novel genetic tools to engineer the interface of primary and specialized metabolism in plants. 

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4. Enabling microbial syringol conversion through structure-guided protein engineering

   https://doi.org/10.1073/pnas.1820001116  

   Machovina, Melodie M.; Mallinson, Sam J.; Knott, Brandon C.; Meyers, Alexander W.; Garcia-Borràs, Marc; Bu, Lintao; Gado, Japheth E.; Oliver, April; Schmidt, Graham P.; Hinchen, Daniel J.; et al (January 2019, Proceedings of the National Academy of Sciences)

   Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O -aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O -demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O -demethylases in the biological conversion of lignin-derived aromatic compounds. 

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5. 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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