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 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 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 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.

Antioxidant potential, anti-nutritional factors, volatile compounds and phenolic composition of microwave heat-treated plum (Prunus domestica. L.) kernels: an analytical approach

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Mohd Aaqib Sheikh ◽  
Charanjiv Singh Saini ◽  
Harish Kumar Sharma
Keyword(s):  
Antioxidant Activity ◽  
Phenolic Compounds ◽  
Microwave Heating ◽  
Treatment Time ◽  
Syringic Acid ◽  
Antioxidant Potential ◽  
Hydrocyanic Acid ◽  
Total Phenolic ◽  
Nutritional Factors ◽  
Content Type

PurposeThe study was aimed to explore the potential impact of microwave heating (450 W for 2, 4, 6 and 8 min) on antioxidant activity, anti-nutritional factors, volatile and phenolic compounds of the plum kernels.Design/methodology/approachPlum kernels are rich in proteins, lipids and bioactive compounds that are mostly underused and undervalued.FindingsThe results showed that microwave heating at 450 W for 6 min significantly (p < 0.05) increased the antioxidant activity, total phenolic and flavonoid content, while the longer treatment time (450 W for 8 min) adversely affected the phenolic compounds. Most importantly, the anti-nutritional factors like amygdalin, hydrocyanic acid, phytic acid and tannin content were reduced up to 87.1, 84.7, 20.9 and 46.2%, respectively at 450 W for 6 min treatment conditions, which was confirmed from the larger shifts observed in FT-IR spectra near 1,157 cm−1. Microwave heating at 450 W for 6 min also proved beneficial in improving the bioavailability of volatile and phenolic compounds including chlorogenic acid, gallic acid, syringic acid, (+)-catechin, caffeic acid, ß-carotene, trans-ferulic acid, rutin trihydrate, 3,4-dihydroxybenzoic acid, tannic acid and quercetin by liberating them from the plant matrix.Originality/valueThe results thus indicate that controlled microwave heating could be an effective approach for the reduction of anti-nutritional factors besides leading to an overall improvement in antioxidant potential and volatile and phenolic compounds. This novel technological approach can proliferate the use of plum kernels in different diversified food formulations.

scholarly journals Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

2015 ◽  
Vol 82 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Daiana de Lima-Morales ◽  
Diego Chaves-Moreno ◽  
Melissa L. Wos-Oxley ◽  
Ruy Jáuregui ◽  
Ramiro Vilchez-Vargas ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Contaminated Site ◽  
Degradation Pathways ◽  
Content Type ◽  
Benzene Degradation ◽  
Semialdehyde Dehydrogenase ◽  
Genes Encoding ◽  
Aromatic Degradation ◽  
Pseudomonas Veronii ◽  
Encoding Gene

ABSTRACTPseudomonas veronii1YdBTEX2, a benzene and toluene degrader, andPseudomonas veronii1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

scholarly journals Comparison of free and bound phenolic compositions and antioxidant activities of leaves from different mulberry varieties

BMC Chemistry ◽  
2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Zhenjiang Wang ◽  
Cuiming Tang ◽  
Gengsheng Xiao ◽  
Fanwei Dai ◽  
Sen Lin ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Phenolic Compounds ◽  
Chlorogenic Acid ◽  
Antioxidant Activities ◽  
Syringic Acid ◽  
Mulberry Leaves ◽  
Phenolic Substances ◽  
Further Development ◽  
Active Substances ◽  
Phenolic Extracts

AbstractMulberry leaves are used in traditional Chinese medicine and contain numerous active substances that are known to be beneficial for human health. The aim of this study was to investigate the phenolic compositions and antioxidant activities of the leaves from 23 mulberry cultivars. Qualitative LC-ESI-QTOF analysis revealed the presence of 11 phenolic compounds in the free phenolic extracts and 10 phenolic compounds in the bound fractions. Chlorogenic acid and caffeic acid were the major components in the free and bound fractions, respectively. The results revealed that the changguosang cultivar from Taiwan contained the greatest content of phenolic compounds as well as the highest antioxidant activity among the 23 cultivars examined, as determined using three separate antioxidant assays. The isoquercitrin, chlorogenic acid, and rutin contents of the free phenolic extracts displayed significant correlations with the antioxidant activities, while syringic acid and rutin were the main contributors to the antioxidant activities of the bound phenolic fractions. The obtained results demonstrate that mulberry leaves contain a variety of beneficial phenolic substances and may be suitable for further development as a herbal medicine.

Effect of the support acidity on the aromatic ring-opening of pyrolysis gasoline over Pt/HZSM-5 catalysts

2009 ◽  
Vol 143 (1-2) ◽  
pp. 115-119 ◽  
Author(s):  
Pedro Castaño ◽  
Alazne Gutiérrez ◽  
Inés Villanueva ◽  
Barbara Pawelec ◽  
Javier Bilbao ◽  
...  
Keyword(s):  
Aromatic Ring ◽  
Ring Opening ◽  
Pyrolysis Gasoline ◽  
Effect Of The Support

scholarly journals Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Maria Dimarogona ◽  
Ioanna Karagiannaki ◽  
Angelina Chalima ◽  
Ayelet Fishman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Fruits And Vegetables ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Content Type ◽  
Polyphenol Oxidases ◽  
Increased Activity ◽  
Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

scholarly journals Efficient Plant Biomass Degradation by Thermophilic Fungus Myceliophthora heterothallica

2012 ◽  
Vol 79 (4) ◽  
pp. 1316-1324 ◽  
Author(s):  
Joost van den Brink ◽  
Gonny C. J. van Muiswinkel ◽  
Bart Theelen ◽  
Sandra W. A. Hinz ◽  
Ronald P. de Vries
Keyword(s):  
Wheat Straw ◽  
Plant Biomass ◽  
Hydrolytic Enzymes ◽  
Reaction Times ◽  
Giant Reed ◽  
Thermophilic Fungi ◽  
Biomass Degradation ◽  
Thermostable Enzymes ◽  
Content Type

ABSTRACTRapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such asTrichodermaandAspergillusspecies. The genusMyceliophthoracontains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging toM. heterothallicawere recently separated from the well-described speciesM. thermophila. We evaluate here the potential ofM. heterothallicaisolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilicMyceliophthoraspecies, isolates belonging toM. heterothallicaandM. thermophilagrew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles andin vitroassays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly betweenM. thermophilaandM. heterothallicaisolates. Compared toM. thermophila,M. heterothallicaisolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures ofMyceliophthoraspecies lack sufficient β-xylosidase activity. Sexual crossing of twoM. heterothallicashowed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures ofM. heterothallica.

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.

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