In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

AbstractThe β-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

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The infection cushion: a fungal “weapon” of plant-biomass destruction

10.1101/2020.06.26.173369 ◽

2020 ◽

Author(s):

Mathias Choquer ◽

Christine Rascle ◽

Isabelle R Gonçalves ◽

Amélie de Vallée ◽

Cécile Ribot ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell Wall ◽

Plant Biomass ◽

Ornamental Plants ◽

Sugar Transport ◽

Differential Staining ◽

List Type ◽

In Planta ◽

Cell Wall Degrading Enzymes

SummaryGrey mold disease affects fruits, vegetables and ornamental plants around the world, causing considerable losses every year. Its causing agent, the necrotrophic fungus Botrytis cinerea, produces infection cushions (IC) that are compound appressorial structures dedicated to the penetration of the plant tissues.A microarray analysis was performed to identify genes up-regulated in mature IC. The expression data were supported by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and mutagenesis of two candidate genes.1,231 up-regulated genes and 79 up-accumulated proteins were identified. They highlight a secretion of ROS, secondary metabolites including phytotoxins, and proteins involved in virulence: proteases, plant cell wall degrading enzymes and necrosis inducers. The role in pathogenesis was confirmed for two up-regulated fasciclin genes. DHN-melanin pathway and chitin deacetylases genes are up-regulated and the conversion of chitin into chitosan was confirmed by differential staining of the IC cell wall. In addition, up-regulation of sugar transport and sugar catabolism encoding genes was found.These results support a role for the B. cinerea IC in plant penetration and suggest other unexpected roles for this fungal organ, in camouflage, necrotrophy or nutrition of the pathogen.

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Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes

Science ◽

10.1126/science.abj1342 ◽

2021 ◽

Vol 373 (6556) ◽

pp. 774-779

Author(s):

Federico Sabbadin ◽

Saioa Urresti ◽

Bernard Henrissat ◽

Anna O. Avrova ◽

Lydia R. J. Welsh ◽

...

Keyword(s):

Plant Cell Wall ◽

Crop Protection ◽

Model Organism ◽

Plant Infection ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases ◽

Encoding Gene ◽

Plant Pathogen Interactions ◽

Encoding Genes ◽

Secreted Enzymes

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans. We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

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Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.01408-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 14

Author(s):

Silvia Hüttner ◽

Anikó Várnai ◽

Dejan M. Petrović ◽

Cao Xuan Bach ◽

Dang Thi Kim Anh ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Functional Characterization ◽

Cellulose Microfibrils ◽

Cell Wall Polysaccharide ◽

Poor Growth ◽

Content Type ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

ABSTRACT The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall. IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

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Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015545 ◽

2020 ◽

pp. jbc.RA120.015545

Author(s):

Kristian E. H. Frandsen ◽

Mireille Haon ◽

Sacha Grisel ◽

Bernard Henrissat ◽

Leila Lo Leggio ◽

...

Keyword(s):

Substrate Specificity ◽

Time Course ◽

Plant Cell Wall ◽

Plant Biomass ◽

Substrate Binding ◽

Glycoside Hydrolases ◽

Sequence Alignments ◽

Lytic Polysaccharide Monooxygenases ◽

Molecular Determinants ◽

Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

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Learning from microbial strategies for polysaccharide degradation

Biochemical Society Transactions ◽

10.1042/bst20150180 ◽

2016 ◽

Vol 44 (1) ◽

pp. 94-108 ◽

Cited By ~ 35

Author(s):

Glyn R. Hemsworth ◽

Guillaume Déjean ◽

Gideon J. Davies ◽

Harry Brumer

Keyword(s):

Energy Source ◽

Plant Cell Wall ◽

Biomass Conversion ◽

Human Gut ◽

Human Diet ◽

Healthy Human ◽

Polysaccharide Degradation ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases ◽

Polysaccharide Utilization Loci

Complex carbohydrates are ubiquitous in all kingdoms of life. As major components of the plant cell wall they constitute both a rich renewable carbon source for biotechnological transformation into fuels, chemicals and materials, and also form an important energy source as part of a healthy human diet. In both contexts, there has been significant, sustained interest in understanding how microbes transform these substrates. Classical perspectives of microbial polysaccharide degradation are currently being augmented by recent advances in the discovery of lytic polysaccharide monooxygenases (LPMOs) and polysaccharide utilization loci (PULs). Fundamental discoveries in carbohydrate enzymology are both advancing biological understanding, as well as informing applications in industrial biomass conversion and modulation of the human gut microbiota to mediate health benefits.

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The dual function receptor kinase, OsWAKL21.2, is involved in elaboration of lipaseA/esterase induced immune responses in rice

10.1101/754234 ◽

2019 ◽

Author(s):

Kamal Kumar Malukani ◽

Ashish Ranjan ◽

Hota Shiva Jyothi ◽

Hitendra Kumar Patel ◽

Ramesh V. Sonti

Keyword(s):

Cell Wall ◽

Immune Responses ◽

Plant Pathogens ◽

Plant Cell Wall ◽

Ectopic Expression ◽

Dual Function ◽

Receptor Kinase ◽

Cell Wall Degrading Enzymes ◽

Cell Wall Damage

AbstractPlant pathogens secrete cell wall degrading enzymes (CWDEs) to degrade various components of the plant cell wall. Plants sense this cell wall damage as a mark of infection and induce immune responses. Little is known about the plant functions that are involved in the elaboration of cell wall damage-induced immune responses. Transcriptome analysis revealed that a rice receptor kinase, WALL-ASSOCIATED KINASE-LIKE 21 (OsWAKL21.2), is upregulated following treatment with either Xanthomonas oryzae pv. oryzae (Xoo, a bacterial pathogen) or lipaseA/esterase (LipA: a CWDE of Xoo). Downregulation of OsWAKL21.2 attenuates LipA mediated immune responses. Overexpression of OsWAKL21.2 in rice mimics LipA treatment mediated induction of immune responses and enhanced expression of defence related genes, indicating it could be involved in the perception of LipA induced cell wall damage in rice. OsWAKL21.2 is a dual function kinase having in-vitro kinase and guanylate cyclase (GC) activities. Ectopic expression of OsWAKL21.2 in Arabidopsis also activates plant immune responses. Interestingly, OsWAKL21.2 needs kinase activity to activate rice immune responses while in Arabidopsis it needs GC activity. Our study reveals a novel receptor kinase involved in elaboration of cell wall damage induced rice immune responses that can activate similar immune responses in two different species via two different mechanisms.One sentence SummaryA novel rice receptor WAKL21 that sense cell wall damage caused by Xanthomonas secreted cell wall degrading enzyme to induce immune responses.

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Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose

Dalton Transactions ◽

10.1039/c9dt04065j ◽

2020 ◽

Vol 49 (11) ◽

pp. 3413-3422 ◽

Cited By ~ 1

Author(s):

Luisa Ciano ◽

Alessandro Paradisi ◽

Glyn R. Hemsworth ◽

Morten Tovborg ◽

Gideon J. Davies ◽

...

Keyword(s):

Epr Spectroscopy ◽

Active Site ◽

Crystalline Cellulose ◽

Cellulose Fibril ◽

Cellulose Substrate ◽

Lytic Polysaccharide Monooxygenases ◽

Natural Cellulose ◽

Polysaccharide Monooxygenases ◽

Spectroscopy Studies

Semi-orientated EPR spectroscopy reveals that lytic polysaccharide monooxygenases interact with their natural cellulose substrate in a specific way, where the copper active site is positioned adjacent to the edge of a crystalline cellulose fibril.

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The Endo-Arabinanase BcAra1 Is a Novel Host-Specific Virulence Factor of the Necrotic Fungal Phytopathogen Botrytis cinerea

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-14-0036-r ◽

2014 ◽

Vol 27 (8) ◽

pp. 781-792 ◽

Cited By ~ 25

Author(s):

Majse Nafisi ◽

Maria Stranne ◽

Lisha Zhang ◽

Jan A. L. van Kan ◽

Yumiko Sakuragi

Keyword(s):

Cell Wall ◽

Botrytis Cinerea ◽

Plant Cell ◽

Plant Pathogens ◽

Plant Cell Wall ◽

High Efficiency ◽

Microbial Infection ◽

Cell Wall Degrading Enzymes ◽

Novel Host

The plant cell wall is one of the first physical interfaces encountered by plant pathogens and consists of polysaccharides, of which arabinan is an important constituent. During infection, the necrotrophic plant pathogen Botrytis cinerea secretes a cocktail of plant cell-wall-degrading enzymes, including endo-arabinanase activity, which carries out the breakdown of arabinan. The roles of arabinan and endo-arabinanases during microbial infection were thus far elusive. In this study, the gene Bcara1 encoding for a novel α-1,5-L-endo-arabinanase was identified and the heterologously expressed BcAra1 protein was shown to hydrolyze linear arabinan with high efficiency whereas little or no activity was observed against the other oligo- and polysaccharides tested. The Bcara1 knockout mutants displayed reduced arabinanase activity in vitro and severe retardation in secondary lesion formation during infection of Arabidopsis leaves. These results indicate that BcAra1 is a novel endo-arabinanase and plays an important role during the infection of Arabidopsis. Interestingly, the level of Bcara1 transcript was considerably lower during the infection of Nicotiana benthamiana compared with Arabidopsis and, consequently, the ΔBcara1 mutants showed the wild-type level of virulence on N. benthamiana leaves. These results support the conclusion that the expression of Bcara1 is host dependent and is a key determinant of the disease outcome.

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Ralstonia solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4918-4923.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4918-4923 ◽

Cited By ~ 62

Author(s):

Julie Tans-Kersten ◽

Yanfen Guan ◽

Caitilyn Allen

Keyword(s):

Cell Wall ◽

Bacterial Wilt ◽

Plant Cell Wall ◽

Genomic Library ◽

Response Regulator ◽

Disease Process ◽

Pectin Methylesterase ◽

Wild Type ◽

Cell Wall Degrading Enzymes

ABSTRACT Ralstonia (Pseudomonas)solanacearum causes bacterial wilt, a serious disease of many crop plants. The pathogen produces several extracellular plant cell wall-degrading enzymes, including polygalacturonases (PGs) and pectin methylesterase (Pme). Pme removes methyl groups from pectin, thereby facilitating subsequent breakdown of this cell wall component by PGs, which are known bacterial wilt virulence factors. R. solanacearum PGs could not degrade 93% methylated pectin unless the substrate was first demethylated by Pme, but as the degree of methylation of the pectin substrate decreased, PG activity increased. Primers derived from a published pme sequence generated an 800-bp DNA probe fragment, which identified Pme-encoding plasmids from a R. solanacearum genomic library. A pmechromosomal mutant had no detectable Pme activity in vitro and no longer grew on 93% methylated pectin as a carbon source. Curiously, the pme mutant, which had no detectable PG activity on highly methylated pectin, was just as virulent as the wild-type strain on tomato, eggplant (aubergine), and tobacco. Since PG activity is required for full virulence, this result suggests that the pectin in these particular hosts may not be highly methylated, or that the breakdown of highly methylated pectin is not a significant factor in the disease process in general. A positive response regulator of PG production called PehR was not required for wild-type Pme production. However, a mutant strain lacking PhcA, which is a global regulator of several virulence genes, produced no detectable Pme activity. Thus,pme expression is directly or indirectly regulated by PhcA but not by PehR.

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Loss of bcbrn1 and bcpks13 in Botrytis cinerea Not Only Blocks Melanization But Also Increases Vegetative Growth and Virulence

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-15-0085-r ◽

2015 ◽

Vol 28 (10) ◽

pp. 1091-1101 ◽

Cited By ~ 14

Author(s):

Chenghua Zhang ◽

Yifan He ◽

Pinkuan Zhu ◽

Lu Chen ◽

Yiwen Wang ◽

...

Keyword(s):

Cell Wall ◽

Botrytis Cinerea ◽

Plant Cell Wall ◽

Hydrolytic Enzyme ◽

Complete Medium ◽

Cell Wall Degrading Enzymes ◽

Mycelium Growth ◽

Necrotrophic Pathogen ◽

Melanin Biosynthesis

Botrytis cinerea is a necrotrophic pathogen that causes gray mold disease in a broad range of plants. Dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix of B. cinerea, but knowledge of the exact role of melanin biosynthesis in this pathogen is unclear. In this study, we characterize two genes in B. cinerea, bcpks13 and bcbrn1, encoding polyketide synthase and tetrahydroxynaphthalene (THN) reductases, respectively, and both have predicted roles in DHN melanin biosynthesis. The ∆bcpks13 and ∆bcbrn1 mutants show white and orange pigmentation, respectively, and the mutants are also deficient in conidiation in vitro but show enhanced growth rates and virulence on hosts. Moreover, the mutants display elevated acidification of the complete medium (CM), probably due to oxalic acid secretion and secretion of cell wall–degrading enzymes, and preferably utilize plant cell-wall components as carbon sources for mycelium growth in vitro. In contrast, overexpression of bcbrn1 (OE::bcbrn1 strain) results in attenuated hydrolytic enzyme secretion, acidification ability, and virulence. Taken together, these results indicate that bcpks13 and bcbrn1 participate in diverse cellular and developmental processes, such as melanization and conidiation in B. cinerea in vitro, but they negatively regulate the virulence of this pathogen.

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