Biochemical studies of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare and their roles in lignocellulose degradation

Abstract Background: Ceriporiopsis subvermispora is a white-rot fungus that displays a high specificity towards lignin mineralization when colonizing dead wood or lignocellulosic compounds. The lignocellulose degrading system from C. subvermispora is formed by genes that encode cellulose hydrolytic enzymes, manganese peroxidases, and laccases that catalyze the efficient depolymerization and mineralization of lignin in the presence of Mn3+ through the formation of lipoperoxides from unsaturated lipid acids. This highly specific lignin-degrading system is unique among white-rot fungi. Methods: In order to determine if this metabolic specialization has modified codon usage of the ligninolytic system, leading to an increased adaptation to the fungal translational machine, we analyzed the adaptation to host codon usage (CAI), tRNA pool (tAI, and AAtAI), codon pair bias (CPB) and the number of effective codons (Nc). These indexes were correlated with gene expression of C. subvermispora, as evaluated by microarray in the presence of two carbon sources, glucose and Aspen wood.Results: General gene expression of C. subvermispora was not correlated with the CAI, tAI, AAtAI, CBP or Nc indexes used to evaluate adaptation to codon bias or the tRNA pool, neither in the presence of glucose or Aspen wood. However, in media containing Aspen wood, the induction of expression of lignin-degrading genes showed a strong correlation with all the former indexes. Lignin-degrading genes, defined as genes whose expression increases at least two-fold in Aspen wood, showed significantly (p<0.001) higher values of CAI, AAtAI, CPB, tAI and lower values of Nc with respect to non-induced genes. Among ligninolytic genes, cellulose-binding proteins and manganese peroxidases presented the highest adaptation values. We also identified an expansion of genes encoding glycine and glutamic acid tRNAs.Conclusions: Our results suggest that the metabolic specialization to use wood as the sole carbon source has introduced a bias in the codon usage of genes involved in lignocellulose degradation. This bias reduces codon diversity and increases codon usage adaptation to the tRNA pool available in C. subvermispora. To our knowledge, this is the first study showing that codon usage is modified to improve the translation efficiency of a group of genes involved in a particular metabolic pathway.

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The Pathogenic White-Rot Fungus Heterobasidion parviporum Responds to Spruce Xylem Defense by Enhanced Production of Oxalic Acid

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-12-0029-r ◽

2012 ◽

Vol 25 (11) ◽

pp. 1450-1458 ◽

Cited By ~ 7

Author(s):

Nina Elisabeth Nagy ◽

Harald Kvaalen ◽

Monica Fongen ◽

Carl Gunnar Fossdal ◽

Nicholas Clarke ◽

...

Keyword(s):

Oxalic Acid ◽

Reaction Zone ◽

Antimicrobial Properties ◽

White Rot Fungi ◽

White Rot ◽

White Rot Fungus ◽

Lignocellulose Degradation ◽

Crystal Formation ◽

Enhanced Production ◽

Fungal Genes

Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

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Lytic Polysaccharide Monooxygenases: The Microbial Power Tool for Lignocellulose Degradation

Trends in Plant Science ◽

10.1016/j.tplants.2016.07.012 ◽

2016 ◽

Vol 21 (11) ◽

pp. 926-936 ◽

Cited By ~ 79

Author(s):

Katja Salomon Johansen

Keyword(s):

Lignocellulose Degradation ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

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Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Biotechnology for Biofuels ◽

10.1186/s13068-020-01860-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

James Li ◽

Laleh Solhi ◽

Ethan D. Goddard-Borger ◽

Yann Mathieu ◽

Warren W. Wakarchuk ◽

...

Keyword(s):

Biochemical Characterization ◽

Morphological Changes ◽

Cellulose Degradation ◽

High Surface ◽

High Surface Area ◽

Hydrolytic Enzymes ◽

Lignocellulose Degradation ◽

Cellulomonas Flavigena ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Abstract Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

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Metabolic Specialization and Codon Preference of Lignocellulolytic Genes in the White Rot Basidiomycete Ceriporiopsis subvermispora

Genes ◽

10.3390/genes11101227 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1227

Author(s):

Alex Gonzalez ◽

Gino Corsini ◽

Sergio Lobos ◽

Daniela Seelenfreund ◽

Mario Tello

Keyword(s):

Gene Expression ◽

Codon Usage ◽

Hydrolytic Enzymes ◽

High Specificity ◽

White Rot ◽

White Rot Fungus ◽

Lignocellulose Degradation ◽

Translation Efficiency ◽

Aspen Wood ◽

Ceriporiopsis Subvermispora

Ceriporiopsis subvermispora is a white-rot fungus with a high specificity towards lignin mineralization when colonizing dead wood or lignocellulosic compounds. Its lignocellulose degrading system is formed by cellulose hydrolytic enzymes, manganese peroxidases, and laccases that catalyze the efficient depolymerization and mineralization of lignocellulose. To determine if this metabolic specialization has modified codon usage of the lignocellulolytic system, improving its adaptation to the fungal translational machine, we analyzed the adaptation to host codon usage (CAI), tRNA pool (tAI, and AAtAI), codon pair bias (CPB), and the number of effective codons (Nc). These indexes were correlated with gene expression of C. subvermispora, in the presence of glucose and Aspen wood. General gene expression was not correlated with the index values. However, in media containing Aspen wood, the induction of expression of lignocellulose-degrading genes, showed significantly (p < 0.001) higher values of CAI, AAtAI, CPB, tAI, and lower values of Nc than non-induced genes. Cellulose-binding proteins and manganese peroxidases presented the highest adaptation values. We also identified an expansion of genes encoding glycine and glutamic acid tRNAs. Our results suggest that the metabolic specialization to use wood as the sole carbon source has introduced a bias in the codon usage of genes involved in lignocellulose degradation. This bias reduces codon diversity and increases codon usage adaptation to the tRNA pool available in C. subvermispora. To our knowledge, this is the first study showing that codon usage is modified to improve the translation efficiency of a group of genes involved in a particular metabolic process.

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OPTIMASI KONDISI EKTRAKSI VANILLIN HASIL DEGRADASI LIGNOSELULOSA BAGAS TEBU MENGGUNAKAN RESPONSE SURFACE METHOD (RSM)

Jurnal Bioteknologi & Biosains Indonesia (JBBI) ◽

10.29122/jbbi.v8i1.4500 ◽

2021 ◽

Vol 8 (1) ◽

pp. 89-104

Author(s):

Irnia Nurika ◽

Faudina Nurin Nisa' ◽

Nurul Azizah ◽

Sri Suhartini

Keyword(s):

Ethyl Acetate ◽

Response Surface ◽

Phanerochaete Chrysosporium ◽

Response Surface Method ◽

Sugarcane Bagasse ◽

Extraction Time ◽

White Rot ◽

White Rot Fungus ◽

Lignocellulose Degradation ◽

Surface Method

Optimization of Vanillin Extraction Conditions from Lignocellulose Degradation of Sugarcane Bagasse using the Response Surface Method (RSM) Sugarcane bagasse is an agricultural waste containing lignocellulose and has the potential to be processed into high-value chemicals such as vanillin. The degradation of sugarcane bagasse lignocellulose can be carried out biologically by the white rot fungus Phanerochaete chrysosporium. This study aims to obtain optimal extraction conditions in the form of ethyl acetate solvent volume and extraction time, using the response surface method (RSM). Two optimized factors were the volume of ethyl acetate (71.72; 80; 100; 120; and 128.28 mL) and the extraction time (35.16; 60; 120; 180; 204.84 minutes). The response variables were the concentration (%) and yield of vanillin (µg g–1). The research on the optimization of the response of vanillin levels and vanillin yield was carried out at 14 days incubation with the highest average total soluble phenol (TSP) value of 0.101 mg g–1. The optimal condition of ethyl acetate volume of 109.730 mL with an extraction time of 137.302 minutes was predicted to produce vanillin levels and yields of 0.0078% and 8.9089 g g–1, respectively, with an accuracy value of 93.4%. Based on the verification results, the optimal vanillin concentration and yield were 0.0077% and 8.9805 g g–1, respectively. Bagas tebu merupakan limbah pertanian yang mengandung lignoselulosa dan berpotensi diolah menjadi bahan kimia bernilai tinggi seperti vanillin. Degradasi lignoselulosa bagas tebu dapat dilakukan secara biologis oleh jamur pelapuk putih Phanerochaete chrysosporium. Penelitian ini bertujuan mendapatkan kondisi ekstraksi optimal berupa volume pelarut etil asetat dan lama waktu ekstraksi, menggunakan response surface method (RSM). Dua faktor yang dioptimasi adalah volume etil asetat (71,72; 80; 100; 120; dan 128,28 mL) dan lama waktu ekstraksi (35,16; 60; 120; 180; 204,84 menit). Variabel respons adalah kadar (%) dan yield vanillin (µg g–1). Penelitian optimasi respons kadar vanillin dan yield vanillin dilakukan pada inkubasi 14 hari dengan nilai total soluble phenol (TSP) rata-rata tertinggi 0,101 mg g–1. Kondisi optimal volume etil asetat 109,730 mL dengan waktu ekstraksi 137,302 menit diprediksi menghasilkan kadar dan yield vanillin sebesar 0,0078% dan 8,9089 µg g–1 dengan nilai ketepatan 93,4%. Berdasar hasil verifikasi, konsentrasi dan yield vanillin yang optimal masing-masing adalah 0,0077% dan 8,9805 µg g–1.

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