[Show abstract][Hide abstract] ABSTRACT: The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.
[Show abstract][Hide abstract] ABSTRACT: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence.
The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in 'fast' transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. 'Slow' transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity.
The elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.
Full-text · Article · Mar 2015 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: Background
Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia.
Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates.
Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to directly convert lignocellulosic substrates to biofuels.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-014-0148-0) contains supplementary material, which is available to authorized users.
Full-text · Article · Dec 2014 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.
Full-text · Article · Apr 2014 · Frontiers in Microbiology
[Show abstract][Hide abstract] ABSTRACT: Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.
[Show abstract][Hide abstract] ABSTRACT: Complete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases. Cellulases that are found in Nature vary considerably in their modular diversity and architecture. They include: non-complexed enzymes with single catalytic domains, independent single peptide chains incorporating multiple catalytic modules, and complexed, scaffolded structures, such as the cellulosome. The discovery of the latter two enzyme architectures has led to a generally held hypothesis that these systems take advantage of intramolecular and intermolecular proximity synergies, respectively, to enhance cellulose degradation. We use domain engineering to exploit both of these concepts to improve cellulase activity relative to the activity of mixtures of the separate catalytic domains.
We show that engineered minicellulosomes can achieve high levels of cellulose conversion on crystalline cellulose by taking advantage of three types of synergism; (1) a complementary synergy produced by interaction of endo- and exo-cellulases, (2) an intramolecular synergy of multiple catalytic modules in a single gene product (this type of synergism being introduced for the first time to minicellulosomes targeting crystalline cellulose), and (3) an intermolecular proximity synergy from the assembly of these cellulases into larger multi-molecular structures called minicellulosomes. The binary minicellulosome constructed in this study consists of an artificial multicatalytic cellulase (CBM4-Ig-GH9-X11-X12-GH8-Doc) and one cellulase with a single catalytic domain (a modified CelS with the structure CBM4-Ig-GH48-Doc), connected by a non-catalytic scaffoldin protein. The high level endo-exo synergy and intramolecular synergies within the artificial multifunctional cellulase have been combined with an additional proximity-dependent synergy produced by incorporation into a minicellulosome demonstrating high conversion of crystalline cellulose (Avicel). Our minicellulosome is the first engineered enzyme system confirmed by test to be capable of both operating at temperatures as high as 60[degree sign]C and converting over 60 % of crystalline cellulose to fermentable sugars.
When compared to previously reported minicellulosomes assembled from cellulases containing only one catalytic module each, our novel minicellulosome demonstrates a method for substantial reduction in the number of peptide chains required, permitting improved heterologous expression of minicellulosomes in microbial hosts. In addition, it has been shown to be capable of substantial conversion of actual crystalline cellulose, as well as of the less-well-ordered and more easily digestible fraction of nominally crystalline cellulose.
Full-text · Article · Aug 2013 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: Nature has evolved multiple enzymatic strategies for the degradation of plant cell wall polysaccharides, which are central to carbon flux in the biosphere and an integral part of renewable biofuels production. Many biomass-degrading organisms secrete synergistic cocktails of individual enzymes with one or several catalytic domains per enzyme, whereas a few bacteria synthesize large multi-enzyme complexes, termed cellulosomes, which contain multiple catalytic units per complex. Both enzyme systems employ similar catalytic chemistries; however, the physical mechanisms by which these enzyme systems degrade polysaccharides are still unclear. Here we examine a prominent example of each type, namely a free-enzyme cocktail expressed by the fungus Hypocrea jecorina and a cellulosome preparation secreted from the anaerobic bacterium Clostridium thermocellum. We observe striking differences in cellulose saccharification exhibited by these systems at the same protein loading. Free enzymes are more active on pretreated biomass and in contrast cellulosomes are much more active on purified cellulose. When combined, these systems display dramatic synergistic enzyme activity on cellulose. To gain further insights, we imaged free enzyme- and cellulosome-digested cellulose and biomass by transmission electron microscopy, which revealed evidence for different mechanisms of cellulose deconstruction by free enzymes and cellulosomes. Specifically, the free enzymes employ an ablative, fibril-sharpening mechanism, whereas cellulosomes physically separate individual cellulose microfibrils from larger particles resulting in enhanced access to cellulose surfaces. Interestingly, when the two enzyme systems are combined, we observe changes to the substrate that suggests mechanisms of synergistic deconstruction. Insight into the different mechanisms underlying these two polysaccharide deconstruction paradigms will eventually enable new strategies for enzyme engineering to overcome biomass recalcitrance.
Full-text · Article · May 2013 · Energy & Environmental Science
[Show abstract][Hide abstract] ABSTRACT: Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical
for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative
imaging in real time to assess the impact of pretreatment, as well as the resulting nanometer-scale changes in cell wall structure,
upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal
cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes
(cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion
is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that
ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially
native microfibrillar structure.
[Show abstract][Hide abstract] ABSTRACT: Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.
[Show abstract][Hide abstract] ABSTRACT: Plant cell walls are composed of three basic structural biomolecules: cellulose, hemicellulose, and lignin with cellulose being the most abundant biopolymer on earth. Cellulose is composed of cellodextrins, which are linear polymers of glucose, and considered to be microcrystalline in structure. The conversion of cellulose to free glucose is one of the primary steps in the fermentative conversion of biomass to fuels and chemicals. However, the crystalline nature of this complex, noncovalent structure is highly resistant to enzymatic hydrolysis. Thus, the substantial cost currently associated with biomass saccharification primarily represents the cost of biomass degrading enzymes. Despite the fact that the microbial cellulose hydrolytic "machinery" for the recycling of carbon from plant biomass already exists in nature, the natural enzymatic degradation of plant material is typically a slow and complex process. Thus, if commercial biofuels production is to become a reality, it must be more cost-effective. One method proposed for achieving this objective is to express all or some of the requisite cellulolytic enzymes in planta, thus reducing both enzyme and thermochemical pretreatment costs.
[Show abstract][Hide abstract] ABSTRACT: Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars.However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process.
In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed.
The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels.
Full-text · Article · Apr 2012 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: The efficient deconstruction of lignocellulosic biomass remains a significant barrier to the commercialization of biofuels. Whereas most commercial plant cell-wall-degrading enzyme preparations used today are derived from fungi, the cellulosomal enzyme system from Clostridium thermocellum is an equally effective catalyst, yet of considerably different structure. A key difference between fungal enzyme systems and cellulosomal enzyme systems is that cellulosomal enzyme systems utilize self-assembled scaffolded multimodule enzymes to deconstruct biomass. Here, the possible function of the X1 modules in the complex multimodular enzyme system cellobiohydrolase A (CbhA) from C. thermocellum is explored. The crystal structures of the two X1 modules from C. thermocellum CbhA have been solved individually and together as one construct. The role that calcium may play in the stability of the X1 modules has also been investigated, as well as the possibility that they interact with each other. Furthermore, the results show that whereas the X1 modules do not seem to act as cellulose disruptors, they do aid in the thermostability of the CbhA complex, effectively allowing it to deconstruct cellulose at a higher temperature.
Full-text · Article · Mar 2012 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: Michael E. Himmel, Gregg T. Beckham, Christina M. Payne, Lintao Bu, James F. Matthews, Stephen R. Decker, John O. Baker, Larry Taylor II, Arjun Singh, Qi Xu, Michael F. Crowley
Biosciences Center, NREL, Golden, CO 80401, USA; firstname.lastname@example.org
The Trichoderma reesei Families 6 and 7 cellulases (Cel6A and Cel7A) are key industrial enzymes used for the production of biofuels from lignocellulosic biomass. These enzymes are multi-modular, with a Family 1 carbohydrate-binding module linked to a large catalytic domain via a flexible O-glycosylated linker. We have used simulation to elucidate new functions for these three sub-domains in general, and have demonstrated a new route to increase the activity of Cel7A. These findings include new roles for glycosylation, which we have shown can be used to tune the binding affinity. We have also examined the structure of the catalytically-active complex of Cel7A and its non-processive counterpart, Cel7B, engaged on cellulose, which suggests allosteric mechanisms may be involved in chain binding when these cellulases are complexed on cellulose. Our computational results also suggest that product inhibition varies significantly between Cel7A and Cel7B, and we offer a molecular-level explanation for this observation. These results highlight new considerations in protein engineering for processive and non-processive cellulases.
Read More: http://informahealthcare.com/doi/full/10.3109/13880209.2012.658722
[Show abstract][Hide abstract] ABSTRACT: Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.
No preview · Article · Mar 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful.
Full-text · Article · May 2010 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate beta-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.
Full-text · Article · Feb 2010 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many
fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process
from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high
amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10,
2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal
stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability
and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120h compared to the heterologously
expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif
at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is
essential for generating engineered enzymes with optimal stability and activity.