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1.
Sci Adv ; 7(26)2021 Jun.
Article in English | MEDLINE | ID: mdl-34172441

ABSTRACT

Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in Saccharomyces cerevisiae that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: We endow full, multifeedstock tolerance on a xylose-consuming strain and one producing the biodegradable plastics precursor lactic acid. The demonstration of "drop-in" hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and nonfuel products alike.

2.
Sci Adv ; 2(2): e1501254, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26989779

ABSTRACT

Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the "cell-free" cellulosomal system. The cell-free cellulosome complex can be seen as a "long range cellulosome" because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions.


Subject(s)
Bacterial Proteins/metabolism , Cellulase/metabolism , Clostridium thermocellum/enzymology , Bacterial Proteins/genetics , Biomass , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cellulase/genetics , Cellulosomes/enzymology , Cellulosomes/ultrastructure , Clostridium thermocellum/genetics , Clostridium thermocellum/ultrastructure , Gene Deletion , Genes, Bacterial , Microscopy, Electron, Transmission , Models, Biological , Proteome/genetics , Proteome/metabolism
4.
Curr Opin Chem Biol ; 29: 100-7, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26529490

ABSTRACT

To improve the deconstruction of biomass, the most abundant terrestrial source of carbon polymers, en route to renewable fuels, chemicals, and materials more knowledge is needed into the mechanistic interplay between thermochemical pretreatment and enzymatic hydrolysis. In this review we highlight recent progress in advanced imaging techniques that have been used to elucidate the effects of thermochemical pretreatment on plant cell walls across a range of spatial scales and the relationship between the substrate structure and the function of various glycoside hydrolase components. The details of substrate and enzyme interactions are not yet fully understood and the challenges of characterizing plant cell wall architecture, how it dictates recalcitrance, and how it relates to enzyme-substrate interactions is the focus for many research groups in the field. Better understanding of how to match pretreatments with improved enzyme mixtures will lead to lower costs for industrial biorefining.


Subject(s)
Cell Wall/metabolism , Lignin/metabolism , Plants/metabolism , Bacteria/enzymology , Bacteria/metabolism , Biocatalysis , Biofuels/analysis , Biofuels/microbiology , Biomass , Cell Wall/chemistry , Cell Wall/enzymology , Cell Wall/ultrastructure , Cellulase/metabolism , Cellulose/chemistry , Cellulose/metabolism , Hydrolysis , Lignin/chemistry , Plants/chemistry , Plants/enzymology , Plants/ultrastructure
5.
FEBS J ; 282(22): 4341-56, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26307003

ABSTRACT

UNLABELLED: Family 1 carbohydrate-binding modules (CBMs) are ubiquitous components of multimodular fungal enzymes that degrade plant cell wall polysaccharides and bind specifically to cellulose. Native glycosylation of family 1 CBMs has been shown to substantially impact multiple physical properties, including thermal and proteolytic stability and cellulose binding affinity. To gain molecular insights into the changes in CBM properties upon glycosylation, solution structures of two glycoforms of a Trichoderma reesei family 1 CBM were studied by NMR spectroscopy: a glycosylated family 1 CBM with a mannose group attached to both Thr1 and Ser3 and a second family 1 CBM with single mannose groups attached to Thr1, Ser3 and Ser14. The structures clearly reveal that monosaccharides at both Ser3 and Ser14 on family 1 CBMs present additional cellulose binding platforms, similar to well-characterized aromatic residues at the binding interface, which align to the cellulose surface. These results are in agreement with previous experimental work demonstrating that glycans at Ser3 and Ser14 impart significant improvements in binding affinity. Additionally, detailed analysis of the NMR structures and molecular simulations indicates that the protein backbone of the CBM is not significantly altered by attachment of monosaccharides, and that the mannose attached to Ser14 may be more flexible than the mannose at Ser3. Overall, the present study reveals how family 1 CBM structures are affected by covalent attachment of monosaccharides, which are likely important post-translational modifications of these common subdomains of fungal plant cell wall degrading enzymes. DATABASE: Structural data have been deposited in the RCSB Protein Data Bank (PDB codes: 2MWJ and 2MWK) and the BioMagRes Bank (BMRB codes: 25331 and 25332) for CBM_M2 and CBM_M3, respectively.


Subject(s)
Cellulose/metabolism , Receptors, Cell Surface/chemistry , Trichoderma/enzymology , Amino Acid Sequence , Glycosylation , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Stability , Solutions
6.
Chem Sci ; 6(12): 7185-7189, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-28966766

ABSTRACT

Protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. Going forward, this study suggests the possibility of designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.

7.
Proc Natl Acad Sci U S A ; 111(21): 7612-7, 2014 May 27.
Article in English | MEDLINE | ID: mdl-24821760

ABSTRACT

The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs.


Subject(s)
Biofuels , Cellulases/metabolism , Lignin/metabolism , Models, Molecular , Receptors, Cell Surface/metabolism , Trichoderma/enzymology , Cellulases/chemistry , Glycosylation , Lignin/chemistry , Molecular Structure , Protein Engineering/methods , Receptors, Cell Surface/chemistry
8.
Science ; 344(6184): 578, 2014 May 09.
Article in English | MEDLINE | ID: mdl-24812382

ABSTRACT

Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.


Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Cellulase/chemistry , Cellulose/chemistry
9.
J Biol Chem ; 289(30): 20960-9, 2014 Jul 25.
Article in English | MEDLINE | ID: mdl-24876380

ABSTRACT

The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding. The hydrophobicity of the enzyme surface was quantified using an estimation of the clustering of nonpolar atoms, identifying potential interaction sites. The adsorption of enzymes to lignin surfaces, measured using the quartz crystal microbalance, correlates to the hydrophobic cluster scores. Further, these results suggest a minimum hydrophobic cluster size for a protein to preferentially adsorb to lignin. The impact of electrostatic contribution was ruled out by comparing the isoelectric point (pI) values to the adsorption of proteins to lignin surfaces. These results demonstrate the ability to predict enzyme-lignin adsorption and could potentially be used to design improved cellulase cocktails, thus lowering the overall cost of biofuel production.


Subject(s)
Aspergillus/enzymology , Fungal Proteins/chemistry , Lignin/chemistry , Oxygenases/chemistry , Adsorption , Hydrophobic and Hydrophilic Interactions , Quartz Crystal Microbalance Techniques
10.
Science ; 342(6165): 1513-6, 2013 Dec 20.
Article in English | MEDLINE | ID: mdl-24357319

ABSTRACT

Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood.


Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Cellulase/chemistry , Cellulose/chemistry , Bacterial Proteins/isolation & purification , Catalysis , Catalytic Domain , Cellulase/isolation & purification , Hot Temperature , Hydrolysis , Substrate Specificity
11.
Proc Natl Acad Sci U S A ; 110(36): 14646-51, 2013 Sep 03.
Article in English | MEDLINE | ID: mdl-23959893

ABSTRACT

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.


Subject(s)
Cellulose 1,4-beta-Cellobiosidase/metabolism , Cellulose/metabolism , Fungal Proteins/metabolism , Lignin/metabolism , Binding Sites , Binding, Competitive , Biocatalysis , Catalytic Domain , Cellulose 1,4-beta-Cellobiosidase/chemistry , Fungal Proteins/chemistry , Glycosylation , Hydrolysis , Mass Spectrometry , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Trichoderma/enzymology , Trichoderma/metabolism
12.
Chembiochem ; 14(4): 445-51, 2013 Mar 04.
Article in English | MEDLINE | ID: mdl-23401343

ABSTRACT

A structure-based computational approach was used to rationally design peptide inhibitors that can target an E3 ligase (SCF(Fbx4) )-substrate (TRF1) interface and subsequent ubiquitylation. Characterization of the inhibitors demonstrates that our sequence-optimization protocol results in an increase in peptide-TRF1 affinity without compromising peptide-protein specificity.


Subject(s)
F-Box Proteins/antagonists & inhibitors , F-Box Proteins/metabolism , Peptides/chemistry , Peptides/pharmacology , Protein Binding/drug effects , Amino Acid Sequence , Drug Design , F-Box Proteins/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Peptides/genetics , Protein Interaction Maps/drug effects , Telomeric Repeat Binding Protein 1/antagonists & inhibitors , Telomeric Repeat Binding Protein 1/chemistry , Telomeric Repeat Binding Protein 1/metabolism , Ubiquitination/drug effects
13.
Proc Natl Acad Sci U S A ; 108(40): 16588-93, 2011 Oct 04.
Article in English | MEDLINE | ID: mdl-21949362

ABSTRACT

Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-containing chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resolution, we find that CENP-A-containing arrays are globally condensed relative to arrays with the canonical histones. At high structural resolution, using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-containing nucleosome structure and higher-order chromatin folding at the centromere.


Subject(s)
Autoantigens/genetics , Centromere/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA, Superhelical/metabolism , Nucleic Acid Conformation , Nucleosomes/genetics , Protein Conformation , Centromere Protein A , Electrophoresis, Polyacrylamide Gel , Epigenomics , Histones/chemistry , Histones/genetics , Humans , Mass Spectrometry , Microscopy, Electron, Transmission , Nucleosomes/ultrastructure , Ultracentrifugation
14.
Biophys J ; 99(11): 3773-81, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21112302

ABSTRACT

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.


Subject(s)
Cellulase/chemistry , Cellulase/metabolism , Trichoderma/enzymology , Amino Acid Sequence , Glycosylation , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Thermodynamics
15.
J Biol Chem ; 284(25): 16716-16722, 2009 Jun 19.
Article in English | MEDLINE | ID: mdl-19395382

ABSTRACT

Mg(2+)-dependent oligomerization of nucleosomal arrays is correlated with higher order folding transitions that stabilize chromosome structure beyond the 30-nm diameter fiber. In the present studies, we have employed a novel mutagenesis-based approach to identify the macromolecular determinants that control H4 N-terminal domain (NTD) function during oligomerization. Core histones were engineered in which 1) the H2A, H2B, and H3 NTDs were swapped onto the H4 histone fold; 2) the length of the H4 NTD and the H2A NTD on the H4 histone fold, were increased; 3) the charge density of the NTDs on the H4 histone fold was increased or decreased; and 4) the H4 NTD was placed on the H2B histone fold. Model nucleosomal arrays were assembled from wild type and mutant core histone octamers, and Mg(2+)-dependent oligomerization was characterized. The results demonstrated that the H2B and H3 NTDs could replace the H4 NTD, as could the H2A NTD if it was duplicated to the length of the native H4 NTD. Arrays oligomerized at lower salt concentrations as the length of the NTD on the H4 histone fold was increased. Mutations that decreased the NTD charge density required more Mg(2+) to oligomerize, whereas mutants that increased the charge density required less salt. Finally, the H4 NTD functioned differently when attached to the H2B histone fold than the H4 histone fold. These studies have revealed new insights into the biochemical basis for H4 NTD effects on genome architecture as well as the protein chemistry that underlies the function of the intrinsically disordered H4 NTD.


Subject(s)
Histones/chemistry , Histones/genetics , Nucleosomes/chemistry , Nucleosomes/genetics , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Amino Acid Sequence , Animals , Histones/metabolism , In Vitro Techniques , Molecular Sequence Data , Mutagenesis , Nucleosomes/metabolism , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolism
16.
Biochem Cell Biol ; 84(4): 411-7, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16936814

ABSTRACT

Chromatin in a eukaryotic nucleus is condensed through 3 hierarchies: primary, secondary, and tertiary chromatin structures. In vitro, when induced with cations, chromatin can self-associate and form large oligomers. This self-association process has been proposed to mimic processes involved in the assembly and maintenance of tertiary chromatin structures in vivo. In this article, we review 30 years of studies of chromatin self-association, with an emphasis on the evidence suggesting that this in vitro process is physiologically relevant.


Subject(s)
Chromatin/metabolism , Genome/physiology , Animals , Cations , Chickens , Chromatin/physiology , Chromosomes/genetics , Chromosomes/physiology , Gene Expression Regulation , Humans , Hydrogen-Ion Concentration
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