The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes

Abstract Background Biomass recalcitrance is governed by various molecular and structural factors but the interplay between these multiscale factors remains unclear. In this study, hot water pretreatment (HWP) was applied to maize stem internodes to highlight the impact of the ultrastructure of the polymers and their interactions on the accessibility and recalcitrance of the lignocellulosic biomass. The impact of HWP was analysed at different scales, from the polymer ultrastructure or water mobility to the cell wall organisation by combining complementary compositional, spectral and NMR analyses. Results HWP increased the kinetics and yield of saccharification. Chemical characterisation showed that HWP altered cell wall composition with a loss of hemicelluloses (up to 45% in the 40-min HWP) and of ferulic acid cross-linking associated with lignin enrichment. The lignin structure was also altered (up to 35% reduction in β–O–4 bonds), associated with slight depolymerisation/repolymerisation depending on the length of treatment. The increase in $${T}_{1\rho }^{H}$$ T 1 ρ H , $${T}_{HH}$$ T HH and specific surface area (SSA) showed that the cellulose environment was looser after pretreatment. These changes were linked to the increased accessibility of more constrained water to the cellulose in the 5–15 nm pore size range. Conclusion The loss of hemicelluloses and changes in polymer structural features caused by HWP led to reorganisation of the lignocellulose matrix. These modifications increased the SSA and redistributed the water thereby increasing the accessibility of cellulases and enhancing hydrolysis. Interestingly, lignin content did not have a negative impact on enzymatic hydrolysis but a higher lignin condensed state appeared to promote saccharification. The environment and organisation of lignin is thus more important than its concentration in explaining cellulose accessibility. Elucidating the interactions between polymers is the key to understanding LB recalcitrance and to identifying the best severity conditions to optimise HWP in sustainable biorefineries.

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The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators

Cells ◽

10.3390/cells10071665 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1665

Author(s):

Natalia Nikonorova ◽

Evan Murphy ◽

Cassio Flavio Fonseca de Lima ◽

Shanshuo Zhu ◽

Brigitte van de Cotte ◽

...

Keyword(s):

Cell Wall ◽

Root Growth ◽

Growth Regulators ◽

Growth Regulation ◽

Phosphorylation Site ◽

Primary Root ◽

Root Tip ◽

Arabidopsis Root ◽

Growth Promoting ◽

The Impact

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

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Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

Applied and Environmental Microbiology ◽

10.1128/aem.00442-08 ◽

2008 ◽

Vol 74 (22) ◽

pp. 6848-6858 ◽

Cited By ~ 78

Author(s):

F. Abram ◽

E. Starr ◽

K. A. G. Karatzas ◽

K. Matlawska-Wasowska ◽

A. Boyd ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Stress Tolerance ◽

Intact Cell ◽

Microscopic Analysis ◽

Phenotypic Characterization ◽

Carbon Utilization ◽

Two Dimensional Gel Electrophoresis ◽

Sigma B ◽

The Impact

ABSTRACT Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

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Spaceflight Induces Novel Regulatory Responses in Arabidopsis Seedling as Revealed by Combined Proteomic and Transcriptomic Analyses

10.21203/rs.2.21481/v2 ◽

2020 ◽

Author(s):

Colin Peter Singer Kruse ◽

Alexander D Meyers ◽

Proma Basu ◽

Sarahann Hutchinson ◽

Darron R Luesse ◽

...

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Membrane Proteins ◽

Plant Growth ◽

Gene Transcription ◽

Space Station ◽

Growth Efficiency ◽

Rna Seq ◽

Plastid Gene ◽

The Impact

Abstract Background: Understanding of gravity sensing and response is critical to long-term human habitation in space and can provide new advantages for terrestrial agriculture. To this end, the altered gene expression profile induced by microgravity has been repeatedly queried by microarray and RNA-seq experiments to understand gravitropism. However, the quantification of altered protein abundance in space has been minimally investigated. Results: Proteomic (iTRAQ-labelled LC-MS/MS) and transcriptomic (RNA-seq) analyses simultaneously quantified protein and transcript differential expression of three-day old, etiolated Arabidopsis thaliana seedlings grown aboard the International Space Station along with their ground control counterparts. Protein extracts were fractionated to isolate soluble and membrane proteins and analyzed to detect differentially phosphorylated peptides. In total, 968 RNAs, 107 soluble proteins, and 103 membrane proteins were identified as differentially expressed. In addition, the proteomic analyses identified 16 differential phosphorylation events. Proteomic data delivered novel insights and simultaneously provided new context to previously made observations of gene expression in microgravity. There is a sweeping shift in post-transcriptional mechanisms of gene regulation including RNA-decapping protein DCP5, the splicing factors GRP7 and GRP8, and AGO4,. These data also indicate AHA2 and FERONIA as well as CESA1 and SHOU4 as central to the cell wall adaptations seen in spaceflight. Patterns of tubulin-a 1, 3,4 and 6 phosphorylation further reveal an interaction of microtubule and redox homeostasis that mirrors osmotic response signaling elements. The absence of gravity also results in a seemingly wasteful dysregulation of plastid gene transcription. Conclusions: The datasets gathered from Arabidopsis seedlings exposed to microgravity revealed marked impacts on post-transcriptional regulation, cell wall synthesis, redox/microtubule dynamics, and plastid gene transcription. The impact of post-transcriptional regulatory alterations represents an unstudied element of the plant microgravity response with the potential to significantly impact plant growth efficiency and beyond. What’s more, addressing the effects of microgravity on AHA2, CESA1, and alpha tubulins has the potential to enhance cytoskeletal organization and cell wall composition, thereby enhancing biomass production and growth in microgravity. Finally, understanding and manipulating the dysregulation of plastid gene transcription has further potential to address the goal of enhancing plant growth in the stressful conditions of microgravity.

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Supercharged Cellulases Show Reduced Non-Productive Binding, But Enhanced Activity, on Pretreated Lignocellulosic Biomass

10.1101/2021.10.17.464688 ◽

2021 ◽

Author(s):

Bhargava Nemmaru ◽

Jenna Douglass ◽

John M Yarbrough ◽

Antonio De Chellis ◽

Srivatsan Shankar ◽

...

Keyword(s):

Cell Wall ◽

Corn Stover ◽

Lignocellulosic Biomass ◽

Hydrolytic Activity ◽

Fine Tuning ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Cell Wall Components ◽

Carbohydrate Binding Modules ◽

Wall Components

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increase hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

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Metabolic Reprogramming in the Opportunistic Yeast Candida albicans in Response to Hypoxia

mSphere ◽

10.1128/msphere.00913-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Anaïs Burgain ◽

Faiza Tebbji ◽

Inès Khemiri ◽

Adnane Sellam

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Metabolic Pathways ◽

Membrane Lipids ◽

Oxygen Depletion ◽

Metabolic Reprogramming ◽

Pathogenic Yeast ◽

Fungal Virulence ◽

Content Type ◽

The Impact

ABSTRACT Hypoxia is the predominant condition that the human opportunistic fungus Candida albicans encounters in the majority of the colonized niches within the host. So far, the impact of such a condition on the overall metabolism of this important human-pathogenic yeast has not been investigated. Here, we have undertaken a time-resolved metabolomics analysis to uncover the metabolic landscape of fungal cells experiencing hypoxia. Our data showed a dynamic reprogramming of many fundamental metabolic pathways, such as glycolysis, the pentose phosphate pathway, and different metabolic routes related to fungal cell wall biogenesis. The C. albicans lipidome was highly affected by oxygen depletion, with an increased level of free fatty acids and biochemical intermediates of membrane lipids, including phospholipids, lysophospholipids, sphingolipids, and mevalonate. The depletion of oxygen-dependent lipids such as ergosterol or phosphatidylcholine with longer and polyunsaturated lateral fatty acid chains was observed only at the later hypoxic time point (180 min). Transcriptomics data supported the main metabolic response to hypoxia when matched to our metabolomic profiles. The hypoxic metabolome reflected different physiological alterations of the cell wall and plasma membrane of C. albicans under an oxygen-limiting environment that were confirmed by different approaches. This study provided a framework for future in vivo investigations to examine relevant hypoxic metabolic trajectories in fungal virulence and fitness within the host. IMPORTANCE A critical aspect of cell fitness is the ability to sense and adapt to variations in oxygen levels in their local environment. Candida albicans is an opportunistic yeast that is the most prevalent human fungal pathogen. While hypoxia is the predominant condition that C. albicans encounters in most of its niches, its impact on fungal metabolism remains unexplored so far. Here, we provided a detailed landscape of the C. albicans metabolome that emphasized the importance of many metabolic routes for the adaptation of this yeast to oxygen depletion. The fungal hypoxic metabolome identified in this work provides a framework for future investigations to assess the contribution of relevant metabolic pathways in the fitness of C. albicans and other human eukaryotic pathogens with similar colonized human niches. As hypoxia is present at most of the fungal infection foci in the host, hypoxic metabolic pathways are thus an attractive target for antifungal therapy.

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DNA nanostructures coordinate gene silencing in mature plants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818290116 ◽

2019 ◽

Vol 116 (15) ◽

pp. 7543-7548 ◽

Cited By ~ 34

Author(s):

Huan Zhang ◽

Gozde S. Demirer ◽

Honglu Zhang ◽

Tianzheng Ye ◽

Natalie S. Goh ◽

...

Keyword(s):

Cell Wall ◽

Gene Silencing ◽

Plant Cell ◽

Mammalian Cells ◽

Plant Cell Wall ◽

Plant Cells ◽

Design Parameters ◽

Dna Nanostructures ◽

Dna Nanostructure ◽

The Impact

Delivery of biomolecules to plants relies onAgrobacteriuminfection or biolistic particle delivery, the former of which is amenable only to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall, which is absent in mammalian cells and poses the dominant physical barrier to biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells; however, nanoparticle-mediated delivery without external mechanical aid remains unexplored for biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver siRNAs, and effectively silence a constitutively expressed gene inNicotiana benthamianaleaves. We show that nanostructure internalization into plant cells and corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures and both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

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The impact of chemical structure on polyphenol bioaccessibility, as a function of processing, cell wall material and pH: A model system

Journal of Food Engineering ◽

10.1016/j.jfoodeng.2020.110304 ◽

2021 ◽

Vol 289 ◽

pp. 110304 ◽

Cited By ~ 2

Author(s):

Eden Eran Nagar ◽

Liora Berenshtein ◽

Inbal Hanuka Katz ◽

Uri Lesmes ◽

Zoya Okun ◽

...

Keyword(s):

Cell Wall ◽

Chemical Structure ◽

Model System ◽

Wall Material ◽

Cell Wall Material ◽

The Impact

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The Cell Envelope Stress Response of Bacillus subtilis towards Laspartomycin C

Antibiotics ◽

10.3390/antibiotics9110729 ◽

2020 ◽

Vol 9 (11) ◽

pp. 729

Author(s):

Angelika Diehl ◽

Thomas M. Wood ◽

Susanne Gebhard ◽

Nathaniel I. Martin ◽

Georg Fritz

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Stress Response ◽

Cell Envelope ◽

Resistance Mechanisms ◽

Detailed Knowledge ◽

Knock Out ◽

Lipid Ii ◽

Envelope Stress ◽

The Impact

Cell wall antibiotics are important tools in our fight against Gram-positive pathogens, but many strains become increasingly resistant against existing drugs. Laspartomycin C is a novel antibiotic that targets undecaprenyl phosphate (UP), a key intermediate in the lipid II cycle of cell wall biosynthesis. While laspartomycin C has been thoroughly examined biochemically, detailed knowledge about potential resistance mechanisms in bacteria is lacking. Here, we use reporter strains to monitor the activity of central resistance modules in the Bacillus subtilis cell envelope stress response network during laspartomycin C attack and determine the impact on the resistance of these modules using knock-out strains. In contrast to the closely related UP-binding antibiotic friulimicin B, which only activates ECF σ factor-controlled stress response modules, we find that laspartomycin C additionally triggers activation of stress response systems reacting to membrane perturbation and blockage of other lipid II cycle intermediates. Interestingly, none of the studied resistance genes conferred any kind of protection against laspartomycin C. While this appears promising for therapeutic use of laspartomycin C, it raises concerns that existing cell envelope stress response networks may already be poised for spontaneous development of resistance during prolonged or repeated exposure to this new antibiotic.

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