scholarly journals Anaerobic degradation of syringic acid by an adapted strain of Rhodopseudomonas palustris

2019 ◽  
Author(s):  
J. Zachary Oshlag ◽  
Yanjun Ma ◽  
Kaitlin Morse ◽  
Brian T. Burger ◽  
Rachelle A. Lemke ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Anaerobic Degradation ◽  
Fossil Fuels ◽  
Plant Biomass ◽  
Rhodopseudomonas Palustris ◽  
Reducing Power ◽  
Sole Source ◽  
Syringic Acid ◽  
Detailed Knowledge ◽  
Acid Degradation

ABSTRACTWhile lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogenous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a byproduct of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions, via the benzoyl-CoA degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619-rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris.IMPORTANCELignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for production of aromatic-based biochemicals requires detailed knowledge of metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveals a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire to metabolize aromatic compounds from lignin and other renewable sources.

scholarly journals Anaerobic Degradation of Syringic Acid by an Adapted Strain of Rhodopseudomonas palustris

2019 ◽  
Vol 86 (3) ◽  
Author(s):  
J. Zachary Oshlag ◽  
Yanjun Ma ◽  
Kaitlin Morse ◽  
Brian T. Burger ◽  
Rachelle A. Lemke ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Anaerobic Degradation ◽  
Fossil Fuels ◽  
Plant Biomass ◽  
Rhodopseudomonas Palustris ◽  
Reducing Power ◽  
Syringic Acid ◽  
Detailed Knowledge ◽  
Content Type ◽  
Acid Degradation

ABSTRACT While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris. IMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

scholarly journals Redundancy in Aromatic O-Demethylation and Ring-Opening Reactions in Novosphingobium aromaticivorans and Their Impact in the Metabolism of Plant-Derived Phenolics

2021 ◽  
Vol 87 (8) ◽  
Author(s):  
Jose M. Perez ◽  
Wayne S. Kontur ◽  
Carson Gehl ◽  
Derek M. Gille ◽  
Yanjun Ma ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Substrate Specificity ◽  
Aromatic Ring ◽  
Ring Opening ◽  
Plant Biomass ◽  
Syringic Acid ◽  
Detailed Knowledge ◽  
Degradation Pathways ◽  
Content Type ◽  
Aromatic Degradation

ABSTRACT Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds, followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM 12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolic compounds by N. aromaticivorans. The results showed the involvement of DesA in O-demethylation of syringic and vanillic acids, LigM in O-demethylation of vanillic acid and 3-MGA, and a new O-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring-opening dioxygenase involved in 3-MGA, PCA, and GA metabolism and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O-demethylation of 3-MGA to GA. We predict that this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

scholarly journals Kinetic modeling of anaerobic degradation of plant-derived aromatic mixtures by Rhodopseudomonas palustris

2021 ◽  
Vol 32 (2) ◽  
pp. 179-192
Author(s):  
Yanjun Ma ◽  
Timothy J. Donohue ◽  
Daniel R. Noguera
Keyword(s):  
Kinetic Modeling ◽  
Vanillic Acid ◽  
Aromatic Compounds ◽  
Rhodopseudomonas Palustris ◽  
Substrate Inhibition ◽  
Carbon Sources ◽  
Syringic Acid ◽  
Hydroxybenzoic Acid ◽  
Organic Substrates ◽  
Substrate Channeling

AbstractRhodopseudomonas 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.

scholarly journals Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

2005 ◽  
Vol 71 (4) ◽  
pp. 2036-2045 ◽  
Author(s):  
Bongkeun Song ◽  
Bess B. Ward
Keyword(s):  
Chesapeake Bay ◽  
Aromatic Compounds ◽  
Anaerobic Degradation ◽  
Coenzyme A ◽  
Sequence Similarity ◽  
Rhodopseudomonas Palustris ◽  
Estuarine Sediment ◽  
Degenerate Primers ◽  
Sediment Samples ◽  
Coa Reductase

ABSTRACT Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.

scholarly journals Functional pathway redundancy in the metabolism of plant-derived phenolics by Novosphingobium aromaticivorans

2020 ◽  
Author(s):  
Jose M. Perez ◽  
Wayne S. Kontur ◽  
Carson Gehl ◽  
Derek M. Gille ◽  
Yanjun Ma ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Substrate Specificity ◽  
Aromatic Ring ◽  
Ring Opening ◽  
Plant Biomass ◽  
Syringic Acid ◽  
Detailed Knowledge ◽  
Degradation Pathways ◽  
Aromatic Degradation ◽  
Functional Pathway

ABSTRACTLignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds by N. aromaticivorans. The results showed the involvement of DesA in O-demethylation of syringic and vanillic acids, LigM in O-demethylation of vanillic acid and 3-MGA, and a new O-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O-demethylation of 3-MGA to GA. We predict this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC.IMPORTANCEFor lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

scholarly journals Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1526
Author(s):  
Joanna E. Kowalczyk ◽  
Shreya Saha ◽  
Miia R. Mäkelä
Keyword(s):  
Genome Editing ◽  
Plant Biomass ◽  
Genetic Manipulation ◽  
White Rot ◽  
White Rot Fungus ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Low Frequencies ◽  
Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

Contributions of Sustainable Biomass and Bioenergy in Agriculture Transitions Towards a Circular Economy

2022 ◽  
pp. 65-80
Author(s):  
Oloiva Maria Tavira ◽  
José Tadeu Marques Aranha ◽  
Maria Raquel Lucas
Keyword(s):  
Climate Change ◽  
Conceptual Model ◽  
Circular Economy ◽  
Fossil Fuels ◽  
Plant Biomass ◽  
Climate Change Impacts ◽  
Use Of Resources ◽  
Starting Point

The production of bioenergy and biofertilizers based on animal and plant biomass is a crucial pillar in circular economy (CE). CE conceptual model and main aims are closely related to the 3 “R” (reduce, reuse, and recycle) rule, which is to improve the use of resources, minimize waste, and assure sustainability. Although bioenergy offers many opportunities and could be an alternative to fossil fuels use, the path for a broader implementation of this type of activity is still long. This study marks the starting point or direction of research to be taken, ensuring the existence of benefits from plant and animal biomass for the production of bioenergy and biofertilizer, as well as the contributions of this type of production to the circular economy and the mitigation of the climate change impacts.

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