scholarly journals A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nikita A. Khlystov ◽  
Yasuo Yoshikuni ◽  
Samuel Deutsch ◽  
Elizabeth S. Sattely
Keyword(s):  
Nicotiana Benthamiana ◽  
Bond Cleavage ◽  
Soluble Form ◽  
Heterologous Host ◽  
Plant Host ◽  
Lignin Valorization ◽  
Degrading Enzymes ◽  
Commodity Chemicals ◽  
Fungal Genes ◽  
Direct Use

AbstractLignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

scholarly journals A plant host enables the heterologous production and combinatorial study of fungal lignin-degrading enzymes

2019 ◽  
Author(s):  
Nikita A. Khlystov ◽  
Yasuo Yoshikuni ◽  
Samuel Deutsch ◽  
Elizabeth S. Sattely
Keyword(s):  
Lignin Degradation ◽  
Bond Cleavage ◽  
Soluble Form ◽  
Heterologous Host ◽  
Plant Host ◽  
Lignin Valorization ◽  
Degrading Enzymes ◽  
Commodity Chemicals ◽  
Fungal Genes ◽  
Direct Use

AbstractLignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

scholarly journals Transcriptome Analysis of Choke Stroma and Asymptomatic Inflorescence Tissues Reveals Changes in Gene Expression in Both Epichloë festucae and Its Host Plant Festuca rubra subsp. rubra

2019 ◽  
Vol 7 (11) ◽  
pp. 567
Author(s):  
Wang ◽  
Clarke ◽  
Belanger
Keyword(s):  
Gene Expression ◽  
Fungal Endophytes ◽  
Plant Stress ◽  
Effector Proteins ◽  
Differentially Expressed ◽  
Festuca Rubra ◽  
Plant Host ◽  
Red Fescue ◽  
Epichloë Festucae ◽  
Fungal Genes

Many cool-season grasses have symbiotic relationships with Epichloë (Ascomycota, Clavicipitaceae) fungal endophytes that inhabit the intercellular spaces of the above-ground parts of the host plants. The presence of the Epichloë endophytes is generally beneficial to the hosts due to enhanced tolerance to biotic and abiotic stresses conferred by the endophytes. Many Epichloë spp. are asexual, and those infections always remain asymptomatic. However, some Epichloë spp. have a sexual stage and produce a macroscopic fruiting body, a stroma, that envelops the developing inflorescence causing a syndrome termed “choke disease”. Here, we report a fungal and plant gene expression analysis of choke stroma tissue and asymptomatic inflorescence tissue of Epichloë festucae-infected strong creeping red fescue (Festuca rubra subsp. rubra). Hundreds of fungal genes and over 10% of the plant genes were differentially expressed when comparing the two tissue types. The differentially expressed fungal genes in the choke stroma tissue indicated a change in carbohydrate and lipid metabolism, as well as a change in expression of numerous genes for candidate effector proteins. Plant stress-related genes were up-regulated in the stroma tissue, suggesting the plant host was responding to the epiphytic stage of E. festucae as a pathogen.

scholarly journals At-CycD2 Enhances Accumulation of Above-Ground Biomass and Recombinant Proteins in Transgenic Nicotiana benthamiana Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Lilya Kopertekh ◽  
Sven Reichardt
Keyword(s):  
Cell Cycle ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Nicotiana Benthamiana ◽  
Production Capacity ◽  
Cell Number ◽  
Phenotypic Characterization ◽  
Above Ground Biomass ◽  
Plant Host ◽  
Ground Biomass

Transient expression in Nicotiana benthamiana holds great potential for recombinant protein manufacturing due to its advantages in terms of speed and yield compared to stably transformed plants. To continue improving the quantity of recombinant proteins the plant host will need to be modified at both plant and cellular levels. In attempt to increase leaf mass fraction, we transformed N. benthamiana with the At-CycD2 gene, a positive regulator of the cell cycle. Phenotypic characterization of the T1 progeny plants revealed their accelerated above-ground biomass accumulation and enhanced rate of leaf initiation. In comparison to non-transgenic control the best performing line At-CycD2-15 provided 143 and 140% higher leaf and stem biomass fractions, respectively. The leaf area enlargement of the At-CycD2-15 genotype was associated with the increase of epidermal cell number compensated by slightly reduced cell size. The production capacity of the At-CycD2-15 transgenic line was superior to that of the non-transgenic N. benthamiana. The accumulation of transiently expressed GFP and scFv-TM43-E10 proteins per unit biomass was increased by 138.5 and 156.7%, respectively, compared to the wild type. With these results we demonstrate the potential of cell cycle regulator gene At-CycD2 to modulate both plant phenotype and intracellular environment of N. benthamiana for enhanced recombinant protein yield.

scholarly journals Transcriptomic Analysis of the White-rot Basidiomycete Lentinus Squarrosulus to Provide Insights Into Its Lignocellulose Biodegradation Ability

2022 ◽  
Author(s):  
Aarthi Ravichandran ◽  
Atul Kolte ◽  
Arindam Dhali ◽  
S Gopinath ◽  
Manpal Srid
Keyword(s):  
Lignocellulosic Biomass ◽  
Ligninolytic Enzymes ◽  
Transcriptomic Analysis ◽  
Glycoside Hydrolases ◽  
White Rot ◽  
Potato Dextrose Broth ◽  
Degrading Enzymes ◽  
Lignocellulose Biodegradation ◽  
Fungal Genes ◽  
Auxiliary Activities

Abstract BackgroundBasidiomycetes are of special interest in biotechnological research for their versatile potential in the degradation of lignocellulosic biomass, chiefly attributed to ligninolytic enzymes along with exo, endo β-glucanases, xylanases, esterases, pectinases, mannanases, cellobiohydrolases, polysaccharide monooxygenases. Relatively little is known about the metabolic process and the subsequent polysaccharide degradation. Transcriptomic analysis of lignicolous fungi grown on different substrates, although attempted by researchers, has focused on a fairly small group of species reporting the expression of fungal genes in response to lignocellulosic biomass as a substrate. This study accordingly reports analysis of transcriptome of a white-rot Basidiomycete L.squarrosulus grown in simple potato dextrose broth supplemented with aromatic compound, reactive black dye to gain an insight into the degradation ability of the fungus. RNA was sequenced using Illumina NextSeq 500 to obtain 6,679,162 high-quality paired-end reads that were assembled de novo using CLC assembly cells to generate 25,244 contigs. Putative functions were assigned for the 10,494 transcripts based on sequence similarities through BLAST2GO 5.2 and Function annotator.ResultsFunctional assignments revealed enhanced oxidoreductase activity through the expression of diverse biomass-degrading enzymes and their corresponding coregulators. CAZyme analysis through dbCAN and CUPP revealed the presence of 6 families of polysaccharide lyases, 51 families of glycoside hydrolases, 23 families of glycoside transferases, 7 families of carbohydrate esterases and 10 families of auxiliary activities. Genes encoding ligninolytic enzymes and auxiliary activities among the transcript sequences were identified through gene prediction by AUGUSTUS and FGENESH. Biochemical analysis of several biomass-degrading enzymes substantiated the functional predictions.ConclusionIn essence, L. squarrosulus grown in a simple medium devoid of lignocellulosic substrate demonstrated the presence of a repertoire of lignocellulose-degrading enzymes, simplying that a source of lignocellulose is not required for the expression of these biomass-degrading enzymes. This study on the transcriptome analysis of L. squarrosulus revealed significant facts on this front and will definitely enhance the knowledge about the biodegradative ability of this fungus, potentially paving the way for efficient biotechnological applications utilizing its potency in biomass degradation and its future functional exploitation in biomass conversion applications.

scholarly journals Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes

2020 ◽  
Vol 71 (10) ◽  
pp. 3052-3065
Author(s):  
Esmer Jongedijk ◽  
Sebastian Müller ◽  
Aalt D J van Dijk ◽  
Elio Schijlen ◽  
Antoine Champagne ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Biosynthetic Pathway ◽  
Glandular Trichomes ◽  
Perillyl Alcohol ◽  
Biosynthesis Pathway ◽  
Geranyl Diphosphate ◽  
Limonene Synthase ◽  
Geranyl Diphosphate Synthase ◽  
Commodity Chemicals ◽  
And Storage

Abstract Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.

scholarly journals Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana

2020 ◽  
Author(s):  
Cuiwei Wang ◽  
Christoph Crocoll ◽  
Niels Agerbirk ◽  
Barbara Ann Halkier
Keyword(s):  
Arabidopsis Thaliana ◽  
Nicotiana Benthamiana ◽  
Biosynthetic Pathway ◽  
Fold Increase ◽  
Chain Elongation ◽  
Heterologous Host ◽  
Barbarea Vulgaris ◽  
Donor Plant ◽  
Defense Compounds ◽  
Novel Approach

AbstractAmong the glucosinolate (GLS) defense compounds characteristic of the Brassicales order, several have been shown to promote human health. This includes 2-phenylethylglucosinolate (2PE) derived from homophenylalanine (HPhe). In this study, we used transient expression in Nicotiana benthamiana to validate and characterize previously predicted key genes in the 2PE biosynthetic pathway from Barbarea vulgaris and demonstrate the feasibility of engineering 2PE production. We used genes from B. vulgaris and Arabidopsis thaliana, in which the biosynthesis of GLSs is predominantly derived from HPhe and dihomomethionine, respectively. The resulting GLS profiles partially mirrored GLS profiles in the gene donor plant, but in both cases the profiles in N. benthamiana were wider than in the native plants. We found that BvBCAT4 is a more efficient entry enzyme for biosynthesis of both HPhe and dihomomethionine and that MAM1 enzymes determine the chain-elongated profile. Co-expression of the chain elongation pathway and CYP79F6 from B. vulgaris with the remaining aliphatic GLS core pathway genes from A. thaliana, demonstrated the feasibility of engineering production of 2PE in N. benthamiana. Noticeably, the HPhe-converting enzyme BvCYP79F6 in the core GLS pathway belongs to the CYP79F subfamily, a family believed to have substrate specificity towards chain-elongated methionine derivatives. Replacing the B. vulgaris chain elongation pathway with a chimeric pathway consisting of BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in an additional 2-fold increase in 2PE production, demonstrating that chimeric pathway with genes from different species can increase flux and boost production in an engineered pathway. Our study provides a novel approach to produce the important HPhe and 2PE in a heterologous host. Chimeric engineering of a complex biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution - and with biotechnological and plant breeding potentials of new-to-nature gene combinations.

scholarly journals Insight into plant cell wall degradation and pathogenesis of Ganoderma boninense via comparative genome analysis

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8065 ◽  
Author(s):  
Ahmad Bazli Ramzi ◽  
Muhammad Lutfi Che Me ◽  
Ummul Syafiqah Ruslan ◽  
Syarul Nataqain Baharum ◽  
Nor Azlan Nor Muhammad
Keyword(s):  
Cell Wall ◽  
Genome Analysis ◽  
Oil Palm ◽  
Pathogenic Fungi ◽  
Comparative Genome Analysis ◽  
Cell Wall Degradation ◽  
Cell Wall Degrading Enzymes ◽  
Plant Host ◽  
Degrading Enzymes ◽  
Host Interaction

Background G. boninense is a hemibiotrophic fungus that infects oil palms (Elaeis guineensis Jacq.) causing basal stem rot (BSR) disease and consequent massive economic losses to the oil palm industry. The pathogenicity of this white-rot fungus has been associated with cell wall degrading enzymes (CWDEs) released during saprophytic and necrotrophic stage of infection of the oil palm host. However, there is a lack of information available on the essentiality of CWDEs in wood-decaying process and pathogenesis of this oil palm pathogen especially at molecular and genome levels. Methods In this study, comparative genome analysis was carried out using the G. boninense NJ3 genome to identify and characterize carbohydrate-active enzyme (CAZymes) including CWDE in the fungal genome. Augustus pipeline was employed for gene identification in G. boninense NJ3 and the produced protein sequences were analyzed via dbCAN pipeline and PhiBase 4.5 database annotation for CAZymes and plant-host interaction (PHI) gene analysis, respectively. Comparison of CAZymes from G. boninense NJ3 was made against G. lucidum, a well-studied model Ganoderma sp. and five selected pathogenic fungi for CAZymes characterization. Functional annotation of PHI genes was carried out using Web Gene Ontology Annotation Plot (WEGO) and was used for selecting candidate PHI genes related to cell wall degradation of G. boninense NJ3. Results G. boninense was enriched with CAZymes and CWDEs in a similar fashion to G. lucidum that corroborate with the lignocellulolytic abilities of both closely-related fungal strains. The role of polysaccharide and cell wall degrading enzymes in the hemibiotrophic mode of infection of G. boninense was investigated by analyzing the fungal CAZymes with necrotrophic Armillaria solidipes, A. mellea, biotrophic Ustilago maydis, Melampsora larici-populina and hemibiotrophic Moniliophthora perniciosa. Profiles of the selected pathogenic fungi demonstrated that necrotizing pathogens including G. boninense NJ3 exhibited an extensive set of CAZymes as compared to the more CAZymes-limited biotrophic pathogens. Following PHI analysis, several candidate genes including polygalacturonase, endo β-1,3-xylanase, β-glucanase and laccase were identified as potential CWDEs that contribute to the plant host interaction and pathogenesis. Discussion This study employed bioinformatics tools for providing a greater understanding of the biological mechanisms underlying the production of CAZymes in G. boninense NJ3. Identification and profiling of the fungal polysaccharide- and lignocellulosic-degrading enzymes would further facilitate in elucidating the infection mechanisms through the production of CWDEs by G. boninense. Identification of CAZymes and CWDE-related PHI genes in G. boninense would serve as the basis for functional studies of genes associated with the fungal virulence and pathogenicity using systems biology and genetic engineering approaches.

Scanning Tunneling Microscopy and Atomic Force Microscopy of Enzymes and Enzyme Complexes

1990 ◽  
Vol 48 (3) ◽  
pp. 174-175
Author(s):  
Ronald D. Edstrom ◽  
Xiuru Yang ◽  
Mary E. Gurnack ◽  
Marcia A. Miller ◽  
Rui Yang ◽  
...  
Keyword(s):  
Cell Biology ◽  
Inorganic Ions ◽  
Bulk Liquid ◽  
Soluble Form ◽  
Scanning Tunneling ◽  
Tunneling Microscopy ◽  
Metabolic Channeling ◽  
Simplistic Notion ◽  
Soluble Enzymes

Many of the questions in biochemistry and cell biology are concerned with the relationships of proteins and other macromolecules in complex arrays which are responsible for carrying out metabolic sequences. The simplistic notion that the enzymes we isolate in soluble form from the cytoplasm were also soluble in vivo is being replaced by the concept that these enzymes occur in organized systems within the cell. In this newer view, the cytoplasm is organized and the “soluble enzymes” are in fact fixed in the cellular space and the only soluble components of the cell are small metabolites, inorganic ions etc. Further support for the concept of metabolic organization is provided by the evidence of metabolic channeling. It has been shown that for some metabolic pathways, the intermediates are not in free diffusion equilibrium with the bulk liquid in the cell but are passed along, more or less directly, from one enzyme to the next.

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