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Challenges and advances in the heterologous expression of cellulolytic enzymes: a review

Biotechnology for Biofuels volume 7, Article number: 135 (2014) Cite this article

Abstract

Second generation biofuel development is increasingly reliant on the recombinant expression of cellulases. Designing or identifying successful expression systems is thus of preeminent importance to industrial progress in the field. Recombinant production of cellulases has been performed using a wide range of expression systems in bacteria, yeasts and plants. In a number of these systems, particularly when using bacteria and plants, significant challenges have been experienced in expressing full-length proteins or proteins at high yield. Further difficulties have been encountered in designing recombinant systems for surface-display of cellulases and for use in consolidated bioprocessing in bacteria and yeast. For establishing cellulase expression in plants, various strategies are utilized to overcome problems, such as the auto-hydrolysis of developing plant cell walls. In this review, we investigate the major challenges, as well as the major advances made to date in the recombinant expression of cellulases across the commonly used bacterial, plant and yeast systems. We review some of the critical aspects to be considered for industrial-scale cellulase production.

Reasons for interest in recombinant cellulases

Cellulases are a crucial component of various industrial processes, such as in cotton and paper manufacturing, as detergent enzymes, in juice extraction and as animal feed additives [1]. Moreover, cellulases are gaining more and more interest for agriculture, biotechnology and bioenergy uses [2], especially in the utilization of cellulosic biomass for the production of renewable liquid biofuels like ethanol, butanol or other fermentative products of sugar. With such uses, cellulases have the potential to become the largest group of industrially-used enzymes worldwide [3].

Cellulases hydrolyze cellulose, one of the three main components of lignocellulose (which also contains lignin and hemicellulose), into sugar molecules. Lignocellulose forms the cell wall and structural tissue of almost all plant systems. It is the most abundantly available regenerative raw material worldwide and, thus, plays an important role as a substrate in the conversion of biomass to biofuels. For the efficient hydrolysis of cellulose, basically three types of synergistically acting enzymes are necessary [4]. Cellobiohydrolases (CBHs), also named exoglucanases, attack the crystalline ends of cellulose producing cellobiose [5]. Endoglucanases (EGs) split glycosidic bonds within the amorphous part of the substrate [6]. Finally, the released cellobiose is cleaved by β-glycosidases (BGls) into glucose monomers [7],[8].

Cellulolytic enzymes are widespread in nature and are found in plants, insects, bacteria and fungi [9]-[11]. Aerobic and anaerobic bacteria are known to produce cellulolytic enzymes as single enzymes or in the form of cellulosomes, multi-enzyme complexes comprising several cellulolytic enzymes bound to a scaffold protein [12]. However, the most common commercially available cellulases so far are non-complexed native enzyme mixtures derived from fungi, especially Trichoderma or Aspergillus species. For use as enzyme producers, cellulolytic fungi have the great advantage of both utilizing secretory pathways and the production of high protein yields [2]. Upwards of 20 g, and reportedly up to 100 g, of crude cellulases per liter are reachable with engineered Trichoderma reesei strains [3],[13]. Additionally, other fungi, such as the genera Penicillium, Acremonium and Chrysosporium are viewed as potential and promising alternatives to Trichoderma[14].

For the conversion of biomass to biofuels on an industrial scale, several obstacles need to be overcome. For example, continued high production costs of cellulases, which comprise up to 20% of the total ethanol production costs as evaluated by the United States National Renewable Energy Laboratory (NREL) [2], minimize production efficiency on a commercial level. Further, to achieve efficient biomass conversion, a set of different enzymes have to act in concert, with the most effective enzyme composition being dependent on the respective biomass substrate [15],[16]. The use of traditional fungal host organisms for cellulase degradation is restricted by the need for special culturing and induction conditions. To overcome these limitations, researchers are not only working on increasing the expression level of cellulolytic fungal cellulases to lower the production costs, but also on the optimization of recombinant expression systems in plants or microorganisms. The latter will allow for creation of microbial strains which express adapted, synergistically active sets of enzymes, either within a single cell [17] or by combining different strains [18], leading to enzyme yields on an economically feasible level.

This review will give an overview of the major problems and challenges of efficient recombinant expression of cellulolytic enzymes. The main focus will be on the differences between the most commonly used host organisms, whether in bacteria, yeast or plants. In particular, focus will be given to the less published challenges encountered, such as cases of enzyme truncation and expression failure of cellulase enzymes, thought to be due to problems occurring during the transport of proteins across the cell wall or intrinsic host-specific mechanisms.

Cell wall features and protein transport of commonly used host organisms

To date, many different host organisms are used for cellulase expression, including bacteria (such as Escherichia and Clostridium species), yeast (such as Saccharomyces and Kluyveromyces species) and plants (such as maize and tobacco). These organisms differ significantly in their cell wall structure, the subcellular compartments for protein targeting and their protein secretion possibilities (Figures 1, 2 and 3).

Figure 1
figure1

Schematic drawing of a bacterial cell showing the localization of recombinantly expressed cellulases and cellulosomes. Recombinant cellulases are expressed and stored in the cytoplasm or targeted to the periplasm through specific protein transporters (such as Tat- and Sec-pathways). Gram-negative bacteria (right) possess a thick outer membrane that restricts extracellular protein transport, thus protein secretion occurs via a specific protein transporter or membrane permeabilization (occurring, for example, due to damage of the cell envelope). Gram-positive bacteria (left) lack the outer membrane leading to a more efficient secretion. Secreted cellulases are either free in solution, either as single cellulases or as cellulosomes, or are displayed on the cell surface via an anchor protein such as Blc or PgsA as single cellulases or as mini-cellulosomes. GH: glycosyl hydrolase; Sec: secretion protein; Tat: twin-arginine-transporter.

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Figure 2
figure2

Illustration of a single yeast cell showing the main cell compartments involved in recombinant protein expression. Arrows indicate the secretory pathway, whereby cellulases are expressed on the ER (1) and transferred via the Golgi apparatus (2) to the medium (3) in secretory vesicles. Cellulases are either free in solution or surface-displayed via an anchor protein (such as α-agglutinin) as single cellulases or as mini-cellulosomes. ER: endoplasmic reticulum; GH: glycosyl hydrolase.

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Figure 3
figure3

Plant cell drawing with the main cell compartments for recombinant protein expression and targeting. Right: Nuclear encoded genes are expressed and targeted to different subcellular compartments using one of two different pathways, indicated by black or grey arrows. On one hand, translation occurs directly into the ER lumen by ribosomes associated with the ER. From here, proteins are transferred into vesicles via the Golgi apparatus (black arrow (1)) to the apoplast (black arrow (2a)) or vacuole (black arrow (2b)) and secreted. Other gene transcripts are translated by free ribosomes (grey arrow (1)) and targeted to the chloroplast (grey arrow (2a)) or mitochondrion (grey arrow (2b)) via specific transit peptides and protein import mechanisms (grey arrows). Left: Enlarged section of the plant cell wall which is primarily composed of cellulose, lignin and hemicellulose. ER: endoplasmic reticulum; TOC/TIC: translocon at the outer envelope of chloroplasts/translocon at the inner envelope of chloroplasts; TOM/TIM: translocase of the outer mitochondrial membrane/translocase of the inner membrane.

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Bacteria are divided into gram-positive and gram-negative bacteria, depending on their cell wall features. Gram-positive bacteria (like Clostridium thermocellum) use protein-secretion mechanisms, including the twin-arginine transporter (Tat) and secretion (Sec) pathways (Figure 1), which export proteins across the cytoplasmic membrane [19]. However, gram-negative bacteria (like Escherichia coli and Zymomonas mobilis) possess an additional outer membrane which restricts the extracellular transport of proteins. Therefore, gram-negative bacteria utilize a variety of secretory pathways for cellulase secretion [20], contributing to the challenges in engineered cellulase production [21].

In contrast to prokaryotic cells, eukaryotic cells are characterized by a nucleus and a variety of organelles. The cells are surrounded by a double lipid layer with a varied composition depending on the organism and its individual function. Yeast cell walls feature a three-layered organization. The inner plasma membrane is covered by an electron transparent layer of glucans, which is complexed with a minor component of chitin, and an outer layer consisting mainly of mannoproteins, which are covalently linked to the inner glucan layer [22]. Similarly to higher eukaryotic cells, protein secretion is directed by an amino-terminal signal peptide sequence which mediates cotranslational translocation into the endoplasmic reticulum (ER), where the signal peptide is removed. N-linked oligosaccharides may be added when the protein is transported via vesicles from the ER to the Golgi, where further modification of these glycosyl structures occurs. From here, the proteins are transferred to the cell surface inside secretory vesicles for secretion (Figure 2).

The plant cell wall differs in composition and thickness according to plant age, species and tissue [15],[23]. A schematic drawing showing the different layers of a plant cell is given in Figure 3. Proteins can accumulate either within the cytoplasm, by targeting via specific signal and transit peptides to different subcellular compartments (the chloroplast, the mitochondria and the ER) or being transported within secretory vesicles from ER through the Golgi network to the vacuole or apoplast [24]. This protein secretion pathway is similar to the above-mentioned yeast secretion pathway, although in plants no hyperglycosylation of proteins occurs [24].

Bacterial expression systems

A wide range of actinomycetes (such as Thermobifida fusca, Thermomonospora curvata and Cellulomonas fimi) [25]-[27], Bacillus species [28] and Clostridia species [29] are cellulase-secreting organisms [20]. Expression of cellulases relies on induction of the relevant genes, therefore these proteins are best expressed when these microbes are grown on cellulosic substrates with no easily metabolizable carbon sources [25]-[29]. The optimal cellulosic inducer varies from organism to organism, and even the most successful induction-associated cellulosic substrates, such as cellobiose and carboxymethyl cellulose (CMC), feature organisms which fail to respond with cellulase production [25]. Not just the carbon source but also the nitrogen and phosphorous sources have been reported to have a significant impact on homologous cellulase expression [27].

Heterologous expression of cellulases can also be triggered in rich growth media by the use of inducible or auto-inducible promoters. For example, the heterologous expression of an alkaline cellulase in a cellulolytic Bacillus strain was achieved under control of a sucrose inducible sacB promoter via the use of a rich growth medium containing yeast extract, tryptone and glucose. Using this system, the expression level of the heterologous cellulase was raised 20-fold, as compared with expression under its own promoter [30]. In another study, a β-galactosidase was expressed in a Bacillus strain under control of an artificial stationary phase specific, auto-inducible promoter, based on an engineered cry3Aa promoter sequence [31], which may also be applicable for cellulase expression. A two-fold increased CelA2 protein production in E. coli was achieved using epMEGAWHOP, a method by which the vector backbone is randomly mutated [32].

The most frequently used bacterial expression host in laboratorial studies is E. coli[33], due to its well-characterized and easy-to-manipulate genetics, the abundance of commercially-available strains and vectors and its ability to express recombinant genes in high yields of up to 50% of the total protein [34]. However, attempts at the expression of cellulases in E. coli have encountered numerous problems such as degradation of linker sequences in multi-domain cellulases [35]-[37], formation of inclusion bodies [38],[39], incorrect transportation across the outer membrane [40] and decreased specific activity of the cellulases [41] (Table 1).

Table 1 Advantages and disadvantages of homologous and heterologous protein expression in different host organisms in regard to technical, ethical and economic aspects
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Further, more general limitations of E. coli as an organism for heterologous cellulase expression are low production levels of soluble proteins and poor secretion ability. Different approaches have been employed to overcome the insolubility of heterologously expressed proteins in E. coli. The soluble expression of previously insoluble cellulases has been achieved in E. coli by strategies such as the fusion of a selected catalytic domain to the carbohydrate-binding module (CBM) of another soluble cellulase [39]. Other strategies are based on the purification of cellulase-containing inclusion bodies using agents such as urea and β-mercaptoethanol with subsequent refolding [42],[43], or on the reduction of the expression temperature to ensure improved protein folding.

One example of temperature-related expression variability in E. coli is the cellulase from the springtail of Cryptopygus antarcticus, which is best expressed as a soluble protein at 10°C [44]. Another potential problem is the low secretion of recombinant cellulases in E. coli, which can be improved by ethylenediaminetetraacetic acid (EDTA) treatment [45] or by the expression of cellulases as OsmY fusion proteins [46]. OsmY is induced by hyperosmotic stress [47] and supports the transport of the fusion proteins over the outer membrane [46], where the fused protein gets cleaved off. Co-expression of the cellulase CelZ and proteins, belonging to the SEC pathways of Erwinia chrysanthemi, facilitate the secretion of CelZ in E. coli[48] and the secretion of E. chrysanthemi EG CleY and CelZ in Klebsiella oxytoca[49], respectively. Further, it was found that the deletion of Braun's lipoprotein makes the outer membrane of E. coli more permeable, which resulted in enhanced secretion of periplasmic proteins and was shown to elevate the overall expression rate of an EG [50].

Another important consideration when expressing cellulases in E. coli is the strain selected for expression. Factors such as whether a strain enables disulfide bridge formation in the cytoplasm (like E. coli Rosetta-gami™) appear to have a significant impact on the expression of certain cellulases [44]. Cellulolytic expression can also be affected by folding factors, with incorrect folding being linked to significant reduction of activity in recombinant proteins compared to their correctly folded counterparts [51]. Lastly, the ongoing optimization of E. coli for recombinant protein expression, for example increasing the ability of secretion via different approaches [34] or the functional transfer of N-linked glycosylation into E. coli[52] is very promising for enhanced cellulase production in this organism since it is assumed that the glycosylation pattern of the linker region of the cellulase is responsible for the flexibility [53] and protease resistance [54] of the enzyme.

Surface-displayed cellulases, chimeric cellulases and cellulosomes

Bacterial expression systems also show great promise in the creation of chimeric cellulases and cellulosomal systems. Chimeric cellulases can be designed to heighten the activities of cellulases by tethering two different activities together [55],[56], however they are more commonly created as components for cellulosomes or mini-cellulosomes [57]-[61]. Bacterial strains used for the expression of these components have included E. coli[62]-[64], but gram-positive Bacillus subtilis or Clostridium species are preferentially used due to their stronger secretion abilities, surface display properties and already being cellulosome-capable [62],[65].

In B. subtilis a number of mini-cellulosomes, or the components to create mini-cellulosomes, have been successfully expressed, containing a wide variety of enzymes, enzyme-binding domains and enzyme-catalytic domains, derived from bacteria such as T. fusca[66],[67] or Clostridium species [62],[68]-[70]. To cite only a few further examples, Clostridium cellulovorans proteins have also been expressed in butanol, acetone and ethanol producing Clostridium acetobutylicum, however with low yields [71]. Mini-cellulosomes with C. thermocellum components have been produced in the industrially-used Lactococcus lactis[72] and in Corynebacterium glutamicum, which can also be used for the production of amino acid products [73]. Cellulosomal components primarily created from cellulase and hemicellulase genes originally found in C. cellulovorans and Clostridium cellulolyticum, but also from such varied sources as Aspergillus niger and Neocallimastix patriciarum, have been successfully expressed in E. coli for in vitro assembly and used either as a complete system [42],[43],[74], in combination with B. subtilis produced components [62] or in systems using nanoparticles as the cellulosome scaffold [63],[64].

The (mini-)cellulosomes described above can be separated into four distinct categories: (1) free in solution, (2) complexed in solution, (3) surface-displayed and (4) mixtures thereof (Figure 1). The critical prerequisite for soluble cellulosomes is that the proteins are properly expressed and secreted. This is not always a simple requirement, as described above. Those enzymes to be complexed in solution require expression of the enzymes of interest, which need to contain a dockerin or other binding module, as well as the expression or presence of a chosen scaffold, such as a protein cohesin or other binding modules [64],[75],[76]. For the production of these heterologous proteins, native secretion mechanisms are most frequently used [73]. The third category, the creation of surface-displayed cellulosomes, requires native secretion or fusion mechanisms as well, however heterologous systems, such as Ruminococcus flavefaciens[69] and Streptococcus pyogenes[72], have also been utilized. Preliminary investigations have been performed examining the surface display of individual cellulase proteins in E. coli, both via lipoproteins, as a proof of principle [77] and via ice nucleation proteins from Pseudomonas syringae for the creation of a screening library to identify improved cellulase variants [78].

Application-related progress using bacteria

Recombinant expression of cellulase enzymes in bacterial systems has proven to be difficult and a number of key challenges remain to be overcome (reviewed in Garvey et al. [79]). However, over the past few years, increasing understanding of the problems faced has led to significant progress in the expression of both single cellulase enzymes and in the creation of cellulase mixtures and cellulosomes in bacteria. There are several reports where a homologous set of cellulases, such as those in B. subtilis, have been supplemented by the introduction of heterologous cellulase-coding sequences [30],[80],[81]. Additionally, non-natively cellulolytic bacteria, like E. coli and Z. mobilis have been transformed into cellulolytic bacteria by genetic manipulation, in order to achieve production of special metabolic products using cellulosic substrates as their carbon source [82],[83] or to enhance the properties of probiotic microorganisms [84]. For example, a carboxymethyl cellulase from the marine B. subtilis was heterologously expressed in E. coli in a 7-liter bioreactor showing a 5.9-fold higher activity rate than in its native organism [85]. Although no clear forerunner for a bacterial consolidated bioprocessing (CBP) organism has yet been established, the tractability of bacteria for protein engineering and transformation into cellulolytic organisms makes this a highly likely prospect for future success [86].

Yeast expression systems

The majority of cellulases are expressed via secretory pathways in their native organisms. When expressed heterologously in E. coli they are, as discussed above, frequently insoluble due to the intrinsic nature and intracellularly-focused expression systems of this bacterium. Yeast, as a host organism, is known for high expression yields in the range of grams per liter [87],[88] and the above-mentioned enzymes are likely to be expressed in soluble and active forms. The secretory pathway of yeasts enables the formation of favored disulfide bonds as well as glycosylation, enhancing the stability and potentially the functionality of the enzymes [89]. Glycosylation of the enzyme linker regions, which typically connect the catalytic domain and the CBM of cellulases, provides protease protection [90] and may also play an important role in assisting the binding of the cellulase to the cellulose surface via the CBM [91]. Further, tractability and easy cultivation conditions are important aspects. This opens the possibility of protein engineering, supported by computational studies (a successful method for cellulase optimization [92]), such as the creation of more active and thermostable enzymes [93].

High throughput approaches, such as flow cytometry and microfluidics-based methods, are also being utilized to identify highly active cellulases, with the potential for use in selecting enzymes from existing or new variant libraries [94]. Another reason for the increasing interest in yeast systems for heterologous cellulase production is their potential for displaying enzymes and enzyme complexes on the surface. Yeast hosts have been demonstrated to be capable of expressing multiple free cellulases within single-cell yeast strains, such as Saccharomyces cerevisiae[95], Saccharomyces pastorianus[96], Pichia pastoris[97],[98] and more recently the Kluyveromyces marxianus[99]. Surface display of cellulase proteins has also been shown in yeast, both for single enzymes [100], multiple enzymes [101] and for (mini-)cellulosome production (see below) (Figure 2). It is notable that, unlike in many bacterial systems, the inherent protein secretion and display mechanisms of yeasts work successfully with cellulase proteins; examples include the use of S. cerevisiae α-mating factor as a secretion signal and α-agglutinin as an anchor protein [82]. Lastly, established tools for genome manipulation and easy cultivation conditions are important aspects for choosing yeasts as an expression host organism.

The expression and secretion of many cellulase-encoding genes in yeast strains have been reported so far and tested on a diversity of substrates, including synthetic compounds as well as amorphous and crystalline forms of cellulose (reviewed in den Haan et al. [102]). Highly efficient secretion was observed by using the yeast α-mating factor for different strains instead of the formerly used yeast signal sequences PHO5 [103] and SUC2 [104]. Overexpression of multiple native SNAREs (soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor proteins), which play an important role during intracellular membrane transport, can increase secretion of heterologously expressed CBH and BGl in S. cerevisiae by up to 52% [105]. Further optimization by random mutagenesis of an EG gene expressed in S. cerevisiae resulted in increased thermostability and extracellular activity [106]. A higher expression yield of CBH was achieved by structure-guided SCHEMA recombination [107] and a 4.5-fold increased Saccharomycopsis fibuligera BGl1 production was realized by the simultaneous overexpression of native S. cerevisiae PSE1 (encoding for a karyopherin) and SOD1 (encoding for a superoxide dismutase). Both proteins were identified through a polygenic analysis for the identification of secretion-enhancing genes [108].

Using δ-integration, a method by which the delta sequence of yeast retrotransposons Ty is used for a multi-copy integration [109], in combination with different auxotrophic markers influences the expression of cellulases significantly, which might be helpful to control the expression of exogenous genes, especially the co-expression of multiple genes [110]. Beside the knowledge of the positive impact of promoter design [111] and (synthetic) terminator regions for mRNA stability and activity [112], an artificial transcriptional activator (TA) was expressed together with an expression cassette containing multiple TA-binding sites and one of three secretory cellulases. An optimal combination of the number of TA-binding sites, the type of core promoter and the terminator region leads to 1.5 to 4-fold increased secretion and 1.5 to 2.5-fold increased activity against phosphoric acid swollen cellulose (PASC) [113]. Looking at other yeast systems, the expression of high yields of foreign cellulases with comparable activity and stability to the native forms were reported in P. pastoris[114]-[116], where a positive effect by codon optimization could be shown [114]. Recently, a P. pastoris strain, based on the commercially available PichiaPink™ expression system, was optimized for the constitutive EG production from Aspergillus at bioreactor scale, which yielded up to 5 g/l total secreted protein [88]. Great progress has also been made for organisms from the species Kluyveromyces[34],[99],[117]. Chang et al. transformed K. marxianus simultaneously with a five-gene cassette, including a CBH, a EG and a BGl, leading to a β-glucan-into-ethanol converting strain [34]. This strain has since been improved by the addition of further cellulases and a cellodextrin transporter gene into the genome, leading to direct conversion of cellulose to ethanol by the recombinant strain [99].

Cell surface display and cellulosomes

The production, secretion and display of cellulolytic proteins as (mini-)cellulosomes in yeast hosts are a significant aim of current research [61]. The generation of such large, complexed cellulase mixtures would help to offset the significantly lower protein expression rates in yeast systems as compared to fungal systems. An economically feasible proposition for future fuel generation is the use of large cellulosomes with significant saccharification capacity to be displayed on a CBP-capable organism [59], with a number of yeasts being viable options. Towards this goal, (mini-)cellulosomes have been developed within S. cerevisiae both as consortia, where enzymes and scaffold proteins are grown in individual strains [118], or as multiple proteins expressed within a single strain [119]. Variations on the use of the α-mating factor secretion pathway were again used to enable successful protein secretion. Attachment mechanisms included α-agglutinin and glycosylphosphatidylinositol anchors [18],[118] as well as systems inherent to the species [119]. A cellulosome consisting of a clostridial scaffoldin, carrying an EG from Nasutitermes takasagoensis was expressed in P. pastoris and linked to the cell surface via a lectin-like anchor flocculation protein from S. cerevisiae[92]. Non-surface associated mini-cellulosomes have also been successfully created via the use of yeast secretion mechanisms, which would provide many benefits of cellulosome activity without the difficulties involved in scaffold attachment (Figure 2) [120].

Challenges and drawbacks in cellulase expression and secretion in yeast

Despite the above-mentioned progress, many challenges still lie ahead for economically viable expression of individual cellulases in yeast systems (Table 1). Factors yet to be determined include the reason why CBHs with high homology are secreted at significantly different levels, and why the co-expression of multiple enzymes in one cell leads to decreasing levels compared to their expression in separate single-cell systems [102]. Different molecular biological approaches to overcome this issue have been inconclusively discussed in the literature, such as promoter design or multi-copy integration of the gene sequence, whether integration is episomal or genomic [96]. Using S. cerevisiae, Ilmen et al. suggested a "compatibility factor", characterized by different levels of plasmid, mRNA and secreted proteins, to explain how some CBHs are more compatible with high level expression and production than others [121]. Variations in the unfolded protein response induction indicate different levels of cellular stress by the expression of different cellulases. This response is suggested to be a novel type of feedback mechanism called repression under secretion stress (RESS) that is activated in response to an impairment of protein folding and secretion. To avoid RESS, the authors recommend using constitutive glycolytic yeast promoters during heterologous expression [102].

During the multi-step cellulase secretory process a number of problems may occur. Yeast proteins which assist in folding and disulfide bond formation differ between organisms, affecting the folding of foreign proteins. Misfolding can result in degradation or retention in the ER and Golgi, respectively [122], and the formation of improper disulfide bonds are one possible reason for the small percentage of active enzyme produced, for example in relation to the secreted fraction in the case of T. reesei CBH1 expressed in P. pastoris[123]. Alternatively, expression of Chaetomium thermophilum CBH3 was produced actively in P. pastoris, suggesting that better expression rates may be achievable when using proteins which consist only of single catalytic domains, rather than the more frequent three-domain proteins (containing a catalytic domain, linker and carbohydrate-binding domain) [124]. Another point which has to be taken into consideration is the specific glycosylation provided by each organism. Expression of a protein in a heterologous system will most likely result in a product with different posttranslational modifications compared with the native enzyme. S. cerevisiae, for instance, lacks the Golgi mannosidase which is present in higher eukaryotes, leading to an elongation of the oligosaccharide chains of the recombinant enzymes by adding further mannose residues [122]. Thus, extensive and branched chains are built up which may add benefits such as protease resistance and thermostability or, alternately, may impair correct protein folding, reactivity and proper secretion.

Application-related progress

One challenge is the creation of yeast organisms combining cellulolytic and fermentative capacities. Previously, strains have been created which could convert cellobiose [125], amorphous cellulose [126] and even crystalline cellulose [121],[127], at least partially, to ethanol. Traditionally, S. cerevisiae was chosen for such approaches, including transformation by cellulase genes and improvement of protein secretion. However, no economically feasible CBP organism has so far been created, despite over a decade of work towards this goal [128],[129]. Direct ethanol fermentation was shown for S. cerevisiae, which co-expresses several surface-displayed cellulases and a cellodextrin transporter, but the ethanol production rates were far behind industrial production levels [130]. More recent approaches utilize alternative yeast organisms with the hope of greater success at establishing an economically viable CBP organism. For example, Kluyveromyces sp. secretes proteins more efficiently and with a less extended glycosylation pattern than S. cerevisiae[131],[132]. This, together with its broad growth temperature and pH ranges, makes it a promising candidate for CBP [99]. As mentioned above, direct conversion of cellulose into ethanol has been shown for a recombinant K. marxianus strain, which expressed and secreted five cellulases and one cellodextrin transporter simultaneously [99]. However, these results affirm the potential of K. marxianus and S. cerevisiae as future CBP strains. Separate hydrolysis and fermentation in situ, another possible approach, is focused on the idea of a two-step approach, a pre-hydrolysis of the cellulose substrate followed by fermentation, which enables optimal conditions for each reaction step [96].

Plant expression systems

The most obvious benefits of cellulase production in plants are low production costs and the ease of achievable scale-up. Both are crucial factors for the economically feasible production of lignocellulosic biofuels due to the high amounts of enzymes needed during the process [133] (Table 1).

Cellulase expression in different plant cell compartments

To achieve efficient cellulase expression in plants, differential targeting of the enzymes is an important strategy towards obtaining high yields and activity. Depending on the (sub)cellular compartment targeted, significant effects on enzyme stability and expression level have been observed [134]. Additionally, the ability of different compartments to allow posttranslational modification of cellulases is an important consideration, as this may impact on the enzymatic activity [135], dependent upon the source of the cellulase expressed (fungal or bacterial). One example of the effects caused by differential targeting is seen with the EG E1 from Acidothermus cellulolyticus, which showed low expression levels in the cytosol of several plant species. Higher yields of this enzyme, up to 16% of total soluble protein (TSP), were achieved when targeted to different subcellular compartments like apoplast, vacuole, ER or mitochondria [136]-[138]. Similar results have been shown for bacterial cellulase Cel5A from Thermotoga maritima. Whereas cytosolic protein expression in tobacco leaves failed, the enzyme accumulates up to 5% of TSP when targeted to the chloroplast, vacuole or apoplast [139]. Even higher amounts, up to 9.3% TSP, could be achieved by the expressing of BglB cellulase from T. maritima in the chloroplast of tobacco using a modified alfalfa RbcsK-1A promoter and a transit peptide [140]. This clearly indicates that the choice of the subcellular compartment plays an important role in heterologous cellulases expression in plant systems.

A second important factor in the expression of cellulase enzymes through plant systems is in the potential for truncation of the enzyme or enzymes. This is also somewhat dependent upon the cellulase localization inside the cell. Again, using the example of EG E1 from A. cellulolyticus, it has been observed that the full-length enzyme is mostly expressed in a truncated but active form, representing only the catalytic domain of the enzyme [134]. The expression of only the catalytic domain of E1 resulted in an increased protein yield, independent of the chosen cell compartments. Evidence even suggests that the truncated catalytic domain of E1 has equal or greater activity to the full-length enzyme [134], making this a beneficial alteration for this specific enzyme. However, for other cellulases this may not be the case, in particular it is known that CBHs need their CBMs in addition to the catalytic domain to achieve effective binding and processivity [141]. One alternative to dealing with the possible truncation of enzymes is to produce single domain enzymes, such as the endocellulase from the hyperthermophilic archeon Sulfolobus solfataricus, which has been successfully expressed in its active form in the ER of tobacco [142].

As an alternative to nuclear genome transformation, the chloroplast is another potential site for heterologous expression with reported yields up to 70% of TSP for a recombinant protein antibiotic [143]. Chloroplast transformation techniques have been utilized to increase the yield of in planta production. Thus, different bacterial cellulase enzymes, predominantly derived from T. fusca, were expressed in active form with yields from 2 to 40% of TSP, mostly without degradation effects [144]-[148]. Consequently, for heterologous expression of full-length bacterial cellulases, the prokaryotic environment of the chloroplast seems to be a very suitable site. Chloroplast transformation provides additional advantages like whole operon expression [149] of multiple genes and transgene containment via maternal inheritance [150],[151].

The ER is another promising subcellular compartment for bacterial cellulases due to its chaperones, which lead to better protein folding and stability [152]. Eukaryotic cellulases expressed in plants are commonly derived from T. reesei, for which ER-targeted CBHs led to truncated but active enzymes, for example in maize [136]. The EGs from A. cellulolyticus and S. solfataricus (discussed above) are other examples for successful cellulase production in the plant ER.

There are indications that targeting to the apoplast may also provide good enzyme yields, but this is countered by possible detrimental effects on plant development, such as decreased growth and reduced cellulose content. To avoid these effects, inducible expression systems can be utilized such as the alcohol inducible promoter system described by Klose et al. [153]. However, recombinant cellulase expression can have far more complicated effects on the growth and development of the plants. Some studies even reported positive effects of heterologous cellulase expression on plant development, such as increased length and width of stems, as shown by Hartati et al. [154] who expressed a poplar cellulase in sengon wood to successfully improve the growth performance of this industrially-used species.

When expressing cellulases in plants, it is important to consider all factors concerning the expression levels and relative activity of the enzyme in relation to its localization within the plant. There is no current forerunner for the `best' compartment for cellulase expression, with the variety of activities within the glycosyl hydrolase family and the differences in processing of enzymes from different sources making such general statements impossible. As a result, the heterologous expression of each enzyme will need to be examined independently for the effects on plant growth and development, as well as for enzyme yield and activity. Despite this, plant systems remain one of the most promising methods for cellulase production on a large and sustainable scale.

Different plant sources for biomass utilization

Beside the research approaches in model systems like tobacco and Arabidopsis thaliana, expression of glycosyl hydrolases and especially cellulases has also been achieved in a variety of other plant species. Crops and woody plants are already implemented in certain industrial processes and are therefore promising candidates as production platforms for biomass-degrading enzymes like cellulases. The three most prominent world crops (maize, rice and wheat) have already been employed in transgenic glycosyl hydrolase production [155]-[157]. An example that is already commercially available is based on the utilization of starch, where a thermostable α-amylase is being expressed in transgenic maize. This maize, called Enogen™, is currently being utilized by Bonanza BioEnergy (Garden City, Kansas, United States) for the production of ethanol from starch [158].

For lignocellulosic fuel production, a transgenic crop producing cellulolytic enzymes to be utilized in an economical process has yet to be created. However, several studies have already described the heterologous expression of cellulolytic enzymes in maize with remarkable success in achieving high expression levels [136],[155],[159]. Amounts of 0.4 kg/ton maize stover are reported for A. cellulolyticus EG E1, and up to 1.8 kg/ton grain for T. reesei CBHI expressed in maize germplasm [138],[159]. Apart from maize, cellulases have been successfully expressed in other crops, like rice [137],[156] and sugar cane [160],[161]. Studies on woody biomass expressing cellulolytic enzymes are rarer than those for crop plants. Nevertheless, successful expression of a recombinant EG has been produced in poplar plants [157],[162]. In these studies, the impact of the recombinant EG on plant growth and development, especially the cell wall, was highlighted.

All approaches developed to date for producing fuel from lignocellulosic sources face a common challenge. The amount of enzyme produced remains significantly lower compared to the amount required for complete biomass degradation [163],[164]. Hence, the possibility of modifying plant cell walls for improved biomass degradation is a promising addition to using plants purely for cellulase production. Here, the concept is that the in planta expression of enzymes leads to modification of the complex plant cell wall structure (Figure 3) and therefore a reduced recalcitrance of plant biomass during degradation for biofuel processing. An example for this strategy is the heterologous expression of glycosyl hydrolases, cellulases and xylanases, which have been shown to improve the hydrolysis rate of transgenic tobacco and maize [47],[165],[166], as demonstrated by Zhang et al., where transgenic maize expressing different biomass-degrading enzymes was utilized for hydrolysis with subsequent fermentation to ethanol, resulting in a 55% improved conversion of lignocellulose to ethanol compared to control plants [166]. For this reason, more recent studies often incorporate detailed information of cellulase effects on plant growth and development, such as the examination of recombinant EG activity on plant cell walls in the poplar studies mentioned above [157],[162]. It must be noted, however, that for field application of these systems, the plant fitness and robustness will have to be considered. In particular, it would need to be determined if resistance to pathogens and herbivorous insects could be altered through such cell-wall modifications [167].

Concluding remarks

The importance of identifying effective systems for recombinant cellulase expression will be one of the key factors in the success of second generation biofuel production. Enzyme production is a critical factor in the economic validation of these systems and a significant stumbling block in establishing systems at the current level of technology. The progress towards better cellulase expression systems, discussed in this review, reveals two major approaches to meeting the challenges within the field. Firstly, many researchers are using systems biology approaches to overcome the difficulties in the heterologous expression of these proteins, such as by the use of different promoters or sequence optimization to increase protein production, or through the use of bacterial or yeast species variants which provide different folding or posttranslational modification options, enabling higher yields of active protein. A second feasible approach to many of the challenges faced has been by moving from multi-domain cellulase expression to expression of single domain proteins (or those active when only containing a single domain), which removes the problem of low yields due to incorrect folding or processing.

While such strategies have progressed our understanding of recombinant cellulase production and have moved the field closer to a viable solution, large challenges remain before cellulase production yields and activities are high enough to be industrially relevant. Work towards meeting this challenge through enzyme surface display or cellulosome production remains far behind the necessary level for such systems to be implemented in the near future. Similarly, the attempts to establish CBP-capable systems continue to progress slowly, without even a clear lead organism in focus. Currently the most promising systems for cellulase production, as evidenced by commercial interest (other than modified homologous systems), are in plant systems for enzyme production. Here, differential targeting provides great promise for successful production of active cellulases, while the already established systems of large-scale plant farming allow for the simple implementation of new systems. However, the current political climate in many countries towards the use of genetically engineered plants is a major drawback for this approach. Unless there is a change in current thinking, bacterial and yeast cellulase production systems may still be the most feasible choices for future biofuel production.

Authors' contributions

CL and MG coordinated the manuscript. CL, MG, JK, DH, HK and UC wrote the manuscript. CL designed the figures. MG drafted the table. RF gave final approval of the version to be published. All authors read and approved the final manuscript.

Abbreviations

BGl:

β-glucosidase

CBH:

Cellobiohydrolase

CBM:

Carbohydrate-binding module

CBP:

Consolidated bioprocessing

CMC:

Carboxymethyl cellulose

EG:

Endoglucanase

ER:

Endoplasmic reticulum

RESS:

Repression under secretion stress

Sec:

Secretion protein

SNAREs:

Soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor proteins

Tat:

Twin-arginine transporter

TSP:

Total soluble protein

References

  1. 1.

    Bhat MK: Cellulases and related enzymes in biotechnology. Biotechnol Adv. 2000, 18: 355-383.

    Google Scholar 

  2. 2.

    Phitsuwan P, Laohakunjit N, Kerdchoechuen O, Kyu KL, Ratanakhanokchai K: Present and potential applications of cellulases in agriculture, biotechnology, and bioenergy. Folia Microbiol (Praha). 2013, 58: 163-176.

    Google Scholar 

  3. 3.

    Wilson DB: Cellulases and biofuels. Curr Opin Biotechnol. 2009, 20: 295-299.

    Google Scholar 

  4. 4.

    Van Dyk JS, Pletschke BI: A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnol Adv. 2012, 30: 1458-1480.

    Google Scholar 

  5. 5.

    Teeri TT: Crystalline cellulose degradation: new insights into the function of cellobiohydrolases. Trends Biotechnol. 1997, 15: 160-167.

    Google Scholar 

  6. 6.

    Medve J, Karlsson J, Lee D, Tjerneld F: Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes. Biotechnol Bioeng. 1998, 59: 621-634.

    Google Scholar 

  7. 7.

    Zhang YHP, Himmel ME, Mielenz JR: Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv. 2006, 24: 452-481.

    Google Scholar 

  8. 8.

    Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev. 2002, 66: 506-577.

    Google Scholar 

  9. 9.

    Watanabe H, Tokuda G: Cellulolytic systems in insects. Annu Rev Entomol. 2010, 55: 609-632.

    Google Scholar 

  10. 10.

    Fischer R, Ostafe R, Twyman RM: Cellulases from insects. Adv Biochem Eng Biotechnol. 2013, 136: 51-64.

    Google Scholar 

  11. 11.

    Duan CJ, Feng JX: Mining metagenomes for novel cellulase genes. Biotechnol Lett. 2010, 32: 1765-1775.

    Google Scholar 

  12. 12.

    Bayer EA, Lamed R, Himmel ME: The potential of cellulases and cellulosomes for cellulosic waste management. Curr Opin Biotechnol. 2007, 18: 237-245.

    Google Scholar 

  13. 13.

    Peterson R, Nevalainen H: Trichoderma reesei RUT-C30 - thirty years of strain improvement. Microbiology. 2012, 158: 58-68.

    Google Scholar 

  14. 14.

    Gusakov AV: Alternatives to Trichoderma reesei in biofuel production. Trends Biotechnol. 2011, 29: 419-425.

    Google Scholar 

  15. 15.

    Chundawat SP, Beckham GT, Himmel ME, Dale BE: Deconstruction of lignocellulosic biomass to fuels and chemicals. Annu Rev Chem Biomol Eng. 2011, 2: 121-145.

    Google Scholar 

  16. 16.

    Blouzard JC, Coutinho PM, Fierobe HP, Henrissat B, Lignon S, Tardif C, Pages S, de Philip P: Modulation of cellulosome composition in Clostridium cellulolyticum: adaptation to the polysaccharide environment revealed by proteomic and carbohydrate-active enzyme analyses. Proteomics. 2010, 10: 541-554.

    Google Scholar 

  17. 17.

    Mazzoli R, Lamberti C, Pessione E: Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies. Trends Biotechnol. 2012, 30: 111-119.

    Google Scholar 

  18. 18.

    Tsai SL, Oh J, Singh S, Chen R, Chen W: Functional assembly of minicellulosomes on the Saccharomyces cerevisiae cell surface for cellulose hydrolysis and ethanol production. Appl Environ Microbiol. 2009, 75: 6087-6093.

    Google Scholar 

  19. 19.

    Desvaux M, Hebraud M, Talon R, Henderson IR: Secretion and subcellular localizations of bacterial proteins: a semantic awareness issue. Trends Microbiol. 2009, 17: 139-145.

    Google Scholar 

  20. 20.

    Yan S, Wu G: Secretory pathway of cellulase: a mini-review. Biotechnol Biofuels. 2013, 6: 177-

    Google Scholar 

  21. 21.

    Jung SK, Parisutham V, Jeong SH, Lee SK: Heterologous expression of plant cell wall degrading enzymes for effective production of cellulosic biofuels. J Biomed Biotechnol. 2012, 2012: 405842-

    Google Scholar 

  22. 22.

    Osumi M: The ultrastructure of yeast: cell wall structure and formation. Micron. 1998, 29: 207-233.

    Google Scholar 

  23. 23.

    Bonawitz ND, Chapple C: The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu Rev Genet. 2010, 44: 337-363.

    Google Scholar 

  24. 24.

    Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R: Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003, 21: 570-578.

    Google Scholar 

  25. 25.

    Stutzenberger FJ: Cellulolytic activity of Thermomonospora curvata: nutritional requirements for cellulase production. Appl Microbiol. 1972, 24: 77-82.

    Google Scholar 

  26. 26.

    Wilson DB: Biochemistry and genetics of actinomycete cellulases. Crit Rev Biotechnol. 1992, 12: 45-63.

    Google Scholar 

  27. 27.

    Lin E, Wilson DB: Regulation of β-1, 4-endoglucanase synthesis in Thermomonospora fusca. Appl Environ Microbiol. 1987, 53: 1352-1357.

    Google Scholar 

  28. 28.

    Kotchoni O, Shonukan O, Gachomo W: Bacillus pumilus BpCRI 6, a promising candidate for cellulase production under conditions of catabolite repression. Afr J Biotechnol. 2003, 2: 140-146.

    Google Scholar 

  29. 29.

    Garcia-Martinez D, Shinmyo A, Madia A, Demain A: Studies on cellulase production by Clostridium thermocellum. Eur J Appl Microbiol. 1980, 9: 189-197.

    Google Scholar 

  30. 30.

    Liu SL, Du K: Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme. Protein Expres Purif. 2012, 83: 164-168.

    Google Scholar 

  31. 31.

    Lee SJ, Pan JG, Park SH, Choi SK: Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis. J Biotechnol. 2010, 149: 16-20.

    Google Scholar 

  32. 32.

    Jakob F, Lehmann C, Martinez R, Schwaneberg U: Increasing protein production by directed vector backbone evolution. AMB Express. 2013, 3: 39-

    Google Scholar 

  33. 33.

    Bill RM: Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments. Front Microbiol. 2014, 5: 85-

    Google Scholar 

  34. 34.

    Chang JJ, Ho CY, Ho FJ, Tsai TY, Ke HM, Wang CH, Chen HL, Shih MC, Huang CC, Li WH: PGASO: A synthetic biology tool for engineering a cellulolytic yeast. Biotechnol Biofuels. 2012, 5: 53-

    Google Scholar 

  35. 35.

    Mittendorf V, Thomson JA: Cloning of an endo-(1 → 4)-β-glucanase gene, celA, from the rumen bacterium Clostridium sp. (`C. longisporum') and characterization of its product, CelA, in Escherichia coli. J Gen Microbiol. 1993, 139: 3233-3242.

    Google Scholar 

  36. 36.

    Meinke A, Gilkes N, Kilburn D, Miller R, Warren R: Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides. J Bacteriol. 1991, 173: 7126-7135.

    Google Scholar 

  37. 37.

    Fierobe HP, Gaudin C, Belaich A, Loutfi M, Faure E, Bagnara C, Baty D, Belaich JP: Characterization of endoglucanase A from Clostridium cellulolyticum. J Bacteriol. 1991, 173: 7956-7962.

    Google Scholar 

  38. 38.

    Jamaluddin MJA, Salleh HM: On-column refolding of recombinant fungal endoglucanase. Aust J Basic and Appl Sci. 2012, 6: 128-133.

    Google Scholar 

  39. 39.

    Murashima K, Kosugi A, Doi RH: Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans. Proteins. 2003, 50: 620-628.

    Google Scholar 

  40. 40.

    Abdeljabbar DM, Song HJ, Link AJ: Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli. Biotechnol Lett. 2012, 34: 91-96.

    Google Scholar 

  41. 41.

    Zhang XZ, Zhang Z, Zhu Z, Sathitsuksanoh N, Yang Y, Zhang YH: The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity. Appl Microbiol Biotechnol. 2010, 86: 525-533.

    Google Scholar 

  42. 42.

    Koukiekolo R, Cho HY, Kosugi A, Inui M, Yukawa H, Doi RH: Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA. Appl Environ Microbiol. 2005, 71: 3504-3511.

    Google Scholar 

  43. 43.

    Mingardon F, Chanal A, Lopez-Contreras AM, Dray C, Bayer EA, Fierobe HP: Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes. Appl Environ Microbiol. 2007, 73: 3822-3832.

    Google Scholar 

  44. 44.

    Song JM, An YJ, Kang MH, Lee YH, Cha SS: Cultivation at 6-10 degrees C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli. Protein Expres Purif. 2012, 82: 297-301.

    Google Scholar 

  45. 45.

    Liu SL, Du K, Chen WZ, Liu G, Xing M: Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme. J Ind Microbiol Biotechnol. 2012, 39: 1301-1307.

    Google Scholar 

  46. 46.

    Gupta S, Adlakha N, Yazdani SS: Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli. Protein Expres Purif. 2013, 88: 20-25.

    Google Scholar 

  47. 47.

    Shen B, Sun X, Zuo X, Shilling T, Apgar J, Ross M, Bougri O, Samoylov V, Parker M, Hancock E, Lucero H, Gray B, Ekborg NA, Zhang D, Johnson JC, Lazar G, Raab RM: Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing. Nat Biotechnol. 2012, 30: 1131-1136.

    Google Scholar 

  48. 48.

    Zhou S, Yomano LP, Saleh AZ, Davis FC, Aldrich HC, Ingram LO: Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B. Appl Environ Microbiol. 1999, 65: 2439-2445.

    Google Scholar 

  49. 49.

    Zhou S, Davis FC, Ingram LO: Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2. Appl Environ Microbiol. 2001, 67: 6-14.

    Google Scholar 

  50. 50.

    Shin HD, Chen RR: Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant. Biotechnol Bioeng. 2008, 101: 1288-1296.

    Google Scholar 

  51. 51.

    Girfoglio M, Rossi M, Cannio R: Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-beta-1-4-glucanase. J Bacteriol. 2012, 194: 5091-5100.

    Google Scholar 

  52. 52.

    Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M: N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science. 2002, 298: 1790-1793.

    Google Scholar 

  53. 53.

    Receveur V, Czjzek M, Schulein M, Panine P, Henrissat B: Dimension, shape, and conformational flexibility of a two domain fungal cellulase in solution probed by small angle X-ray scattering. J Biol Chem. 2002, 277: 40887-40892.

    Google Scholar 

  54. 54.

    Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, Packer NH: Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei. Eur J Biochem. 1998, 256: 119-127.

    Google Scholar 

  55. 55.

    Adlakha N, Rajagopal R, Kumar S, Reddy VS, Yazdani SS: Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Appl Environ Microbiol. 2011, 77: 4859-4866.

    Google Scholar 

  56. 56.

    Adlakha N, Sawant S, Anil A, Lali A, Yazdani SS: Specific fusion of beta-1,4-endoglucanase and beta-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates. Appl Environ Microbiol. 2012, 78: 7447-7454.

    Google Scholar 

  57. 57.

    Fierobe HP, Bayer EA, Tardif C, Czjzek M, Mechaly A, Belaich A, Lamed R, Shoham Y, Belaich JP: Degradation of cellulose substrates by cellulosome chimeras - substrate targeting versus proximity of enzyme components. J Biol Chem. 2002, 277: 49621-49630.

    Google Scholar 

  58. 58.

    Morais S, Barak Y, Lamed R, Wilson DB, Xu Q, Himmel ME, Bayer EA: Paradigmatic status of an endo- and exoglucanase and its effect on crystalline cellulose degradation. Biotechnol Biofuels. 2012, 5: 78-

    Google Scholar 

  59. 59.

    Fontes C, Gilbert HJ: Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates. Annu Rev Biochem. 2010, 79: 655-681.

    Google Scholar 

  60. 60.

    Xu Q, Ding SY, Brunecky R, Bomble YJ, Himmel ME, Baker JO: Improving activity of minicellulosomes by integration of intra- and intermolecular synergies. Biotechnol Biofuels. 2013, 6: 126-

    Google Scholar 

  61. 61.

    Huang GL, Anderson TD, Clubb RT: Engineering microbial surfaces to degrade lignocellulosic biomass. Bioengineered. 2014, 5: 96-106.

    Google Scholar 

  62. 62.

    Cha J, Matsuoka S, Chan H, Yukawa H, Inui M, Doi RH: Effect of multiple copies of cohesins on cellulase and hemicellulase activities of Clostridium cellulovorans mini-cellulosomes. J Microbiol Biotechnol. 2007, 17: 1782-1788.

    Google Scholar 

  63. 63.

    Do-Myoung K, Umetsu M, Takai K, Matsuyama T, Ishida N, Takahashi H, Asano R, Kumagai I: Enhancement of cellulolytic enzyme activity by clustering cellulose binding domains on nanoscaffolds. Small. 2011, 7: 656-664.

    Google Scholar 

  64. 64.

    Kim DM, Nakazawa H, Umetsu M, Matsuyama T, Ishida N, Ikeuchi A, Takahashi H, Asano R, Kumagai I: A nanocluster design for the construction of artificial cellulosomes. Catal Sci Technol. 2012, 2: 499-503.

    Google Scholar 

  65. 65.

    Elkins JG, Raman B, Keller M: Engineered microbial systems for enhanced conversion of lignocellulosic biomass. Curr Opin Biotechnol. 2010, 21: 657-662.

    Google Scholar 

  66. 66.

    Caspi J, Barak Y, Haimovitz R, Irwin D, Lamed R, Wilson DB, Bayer EA: Effect of linker length and dockerin position on conversion of a Thermobifida fusca endoglucanase to the cellulosomal mode. Appl Environ Microbiol. 2009, 75: 7335-7342.

    Google Scholar 

  67. 67.

    Caspi J, Irwin D, Lamed R, Li Y, Fierobe HP, Wilson DB, Bayer EA: Conversion of Thermobifida fusca free exoglucanases into cellulosomal components: comparative impact on cellulose-degrading activity. J Biotechnol. 2008, 135: 351-357.

    Google Scholar 

  68. 68.

    Anderson TD, Miller JI, Fierobe HP, Clubb RT: Recombinant Bacillus subtilis that grows on untreated plant biomass. Appl Environ Microbiol. 2013, 79: 867-876.

    Google Scholar 

  69. 69.

    Anderson TD, Robson SA, Jiang XW, Malmirchegini GR, Fierobe HP, Lazazzera BA, Clubb RT: Assembly of minicellulosomes on the surface of Bacillus subtilis. Appl Environ Microbiol. 2011, 77: 4849-4858.

    Google Scholar 

  70. 70.

    Matsuoka S, Yukawa H, Inui M, Doi RH: Synergistic interaction of Clostridium cellulovorans cellulosomal cellulases and HbpA. J Bacteriol. 2007, 189: 7190-7194.

    Google Scholar 

  71. 71.

    Mingardon F, Chanal A, Tardif C, Fierobe HP: The issue of secretion in heterologous expression of Clostridium cellulolyticum cellulase-encoding genes in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 2011, 77: 2831-2838.

    Google Scholar 

  72. 72.

    Wieczorek AS, Martin VJJ: Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis. Microb Cell Fact. 2010, 9: 89-

    Google Scholar 

  73. 73.

    Hyeon JE, Jeon WJ, Whang SY, Han SO: Production of minicellulosomes for the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium glutamicum. Enzyme Microb Technol. 2011, 48: 371-377.

    Google Scholar 

  74. 74.

    Fierobe HP, Mechaly A, Tardif C, Belaich A, Lamed R, Shoham Y, Belaich JP, Bayer EA: Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes. J Biol Chem. 2001, 276: 21257-21261.

    Google Scholar 

  75. 75.

    Mitsuzawa S, Kagawa H, Li Y, Chan SL, Paavola CD, Trent JD: The rosettazyme: a synthetic cellulosome. J Biotechnol. 2009, 143: 139-144.

    Google Scholar 

  76. 76.

    Morais S, Heyman A, Barak Y, Caspi J, Wilson DB, Lamed R, Shoseyov O, Bayer EA: Enhanced cellulose degradation by nano-complexed enzymes: synergism between a scaffold-linked exoglucanase and a free endoglucanase. J Biotechnol. 2010, 147: 205-211.

    Google Scholar 

  77. 77.

    Francisco JA, Stathopoulos C, Warren RA, Kilburn DG, Georgiou G: Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains. Biotechnol. 1993, 11: 491-495.

    Google Scholar 

  78. 78.

    Kim YS, Jung HC, Pan JG: Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Appl Environ Microbiol. 2000, 66: 788-793.

    Google Scholar 

  79. 79.

    Garvey M, Klose H, Fischer R, Lambertz C, Commandeur U: Cellulases for biomass degradation: comparing recombinant cellulase expression platforms. Trends Biotechnol. 2013, 31: 581-593.

    Google Scholar 

  80. 80.

    Manabe K, Kageyama Y, Morimoto T, Shimizu E, Takahashi H, Kanaya S, Ara K, Ozaki K, Ogasawara N: Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions. Microb Cell Fact. 2013, 12: 18-

    Google Scholar 

  81. 81.

    Liu JM, Xin XJ, Li CX, Xu JH, Bao J: Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis. Appl Biochem Biotechnol. 2012, 166: 652-662.

    Google Scholar 

  82. 82.

    Yamada R, Hasunuma T, Kondo A: Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing. Biotechnol Adv. 2013, 31: 754-763.

    Google Scholar 

  83. 83.

    Bokinsky G, Peralta-Yahya PP, George A, Holmes BM, Steen EJ, Dietrich J, Soon Lee T, Tullman-Ercek D, Voigt CA, Simmons BA, Keasling JD: Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A. 2011, 108: 19949-19954.

    Google Scholar 

  84. 84.

    Liu J-R, Yu B, Liu F-H, Cheng K-J, Zhao X: Expression of rumen microbial fibrolytic enzyme genes in probiotic Lactobacillus reuteri. Appl Environ Microbiol. 2005, 71: 6769-6775.

    Google Scholar 

  85. 85.

    Lee EJ, Lee BH, Kim BK, Lee JW: Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran. Mol Biol Rep. 2013, 40: 3609-3621.

    Google Scholar 

  86. 86.

    la Grange DC, den Haan R, van Zyl WH: Engineering cellulolytic ability into bioprocessing organisms. Appl Microbiol Biotechnol. 2010, 87: 1195-1208.

    Google Scholar 

  87. 87.

    Young CL, Robinson AS: Protein folding and secretion: mechanistic insights advancing recombinant protein production in S. cerevisiae. Curr Opin Biotechnol. 2014, 30C: 168-177.

    Google Scholar 

  88. 88.

    Varnai A, Tang C, Bengtsson O, Atterton A, Mathiesen G, Eijsink VG: Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Microb Cell Fact. 2014, 13: 57-

    Google Scholar 

  89. 89.

    Jeoh T, Michener W, Himmel ME, Decker SR, Adney WS: Implications of cellobiohydrolase glycosylation for use in biomass conversion. Biotechnol Biofuels. 2008, 1: 10-

    Google Scholar 

  90. 90.

    Shen H, Schmuck M, Pilz I, Gilkes NR, Kilburn DG, Miller RC, Warren RA: Deletion of the linker connecting the catalytic and cellulose-binding domains of endoglucanase A (CenA) of Cellulomonas fimi alters its conformation and catalytic activity. J Biol Chem. 1991, 266: 11335-11340.

    Google Scholar 

  91. 91.

    Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE, Sandgren M, Stahlberg J, Stals I, Tan Z, Beckham GT: Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci U S A. 2013, 110: 14646-14651.

    Google Scholar 

  92. 92.

    Bommarius AS, Sohn M, Kang Y, Lee JH, Realff MJ: Protein engineering of cellulases. Curr Opin Biotechnol. 2014, 29C: 139-145.

    Google Scholar 

  93. 93.

    Trudeau DL, Lee TM, Arnold FH: Engineered thermostable fungal cellulases exhibit efficient synergistic cellulose hydrolysis at elevated temperatures.Biotechnol Bioeng 2014. doi:10.1002/bit.25308.,

  94. 94.

    Ostafe R, Prodanovic R, Commandeur U, Fischer R: Flow cytometry-based ultra-high-throughput screening assay for cellulase activity. Anal Biochem. 2013, 435: 93-98.

    Google Scholar 

  95. 95.

    Haan RD, Mcbride JE, la Grange DC, Lynd LR, van Zyl JM: Functional expression of cellobiohydrolases in Saccharomyces cerevisiae. Enzyme Microb Technol. 2007, 40: 1291-1299.

    Google Scholar 

  96. 96.

    Fitzpatrick J, Kricka W, James TC, Bond U: Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose. J Appl Microbiol. 2014, 117: 96-108.

    Google Scholar 

  97. 97.

    Banerjee G, Car S, Scott-Craig JS, Borrusch MS, Aslam N, Walton JD: Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set. Biotechnol Bioeng. 2010, 106: 707-720.

    Google Scholar 

  98. 98.

    Chen P, Fu X, Ng TB, Ye XY: Expression of a secretory beta-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization. Biotechnol Lett. 2011, 33: 2475-2479.

    Google Scholar 

  99. 99.

    Chang JJ, Ho FJ, Ho CY, Wu YC, Hou YH, Huang CC, Shih MC, Li WH: Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production. Biotechnol Biofuels. 2013, 6: 19-

    Google Scholar 

  100. 100.

    Ito J, Kosugi A, Tanaka T, Kuroda K, Shibasaki S, Ogino C, Ueda M, Fukuda H, Doi RH, Kondo A: Regulation of the display ratio of enzymes on the Saccharomyces cerevisiae cell surface by the immunoglobulin G and cellulosomal enzyme binding domains. Appl Environ Microbiol. 2009, 75: 4149-4154.

    Google Scholar 

  101. 101.

    Apiwatanapiwat W, Murata Y, Kosugi A, Yamada R, Kondo A, Arai T, Rugthaworn P, Mori Y: Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and beta-glucosidase. Appl Microbiol Biotechnol. 2011, 90: 377-384.

    Google Scholar 

  102. 102.

    den Haan R, Kroukamp H, van Zyl JHD, van Zyl WH: Cellobiohydrolase secretion by yeast: current state and prospects for improvement. Process Biochem. 2013, 48: 1-12.

    Google Scholar 

  103. 103.

    Arima K, Oshima T, Kubota I, Nakamura N, Mizunaga T, Toh-e A: The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide. Nucleic Acids Res. 1983, 11: 1657-1672.

    Google Scholar 

  104. 104.

    Perlman D, Halvorson HO, Cannon LE: Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence. Proc Natl Acad Sci U S A. 1982, 79: 781-785.

    Google Scholar 

  105. 105.

    Van Zyl JH, Den Haan R, Van Zyl WH: Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion. Appl Microbiol Biotechnol. 2014, 98: 5567-5578.

    Google Scholar 

  106. 106.

    du Plessis L, Rose SH, van Zyl WH: Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains. Appl Microbiol Biotechnol. 2010, 86: 1503-1511.

    Google Scholar 

  107. 107.

    Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A, Govindarajan S, Minshull J, Arnold FH: A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci U S A. 2009, 106: 5610-5615.

    Google Scholar 

  108. 108.

    Kroukamp H, den Haan R, van Wyk N, van Zyl WH: Overexpression of native PSE1 and SOD1 in Saccharomyces cerevisiae improved heterologous cellulase secretion. Appl Energ. 2013, 102: 150-156.

    Google Scholar 

  109. 109.

    Sakai A, Shimizu Y, Hishinuma F: Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty. Appl Microbiol Biotechnol. 1990, 33: 302-306.

    Google Scholar 

  110. 110.

    Liu L, Liu C, Zou S, Yang H, Hong J, Ma Y, Zhang M: Expression of cellulase genes in Saccharomyces cerevisiae via delta-integration subject to auxotrophic markers. Biotechnol Lett. 2013, 35: 1303-1307.

    Google Scholar 

  111. 111.

    Blazeck J, Alper HS: Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J. 2013, 8: 46-58.

    Google Scholar 

  112. 112.

    Yamanishi M, Ito Y, Kintaka R, Imamura C, Katahira S, Ikeuchi A, Moriya H, Matsuyama T: A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a "terminatome" toolbox. ACS Synth Biol. 2013, 2: 337-347.

    Google Scholar 

  113. 113.

    Ito Y, Yamanishi M, Ikeuchi A, Matsuyama T: A highly tunable system for the simultaneous expression of multiple enzymes inSaccharomyces cerevisiae.ACS Synth Biol 2014. doi:10.1021/sb500096y.,

  114. 114.

    Akcapinar GB, Gul O, Sezerman U: Effect of codon optimization on the expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris. Biotechnol Prog. 2011, 27: 1257-1263.

    Google Scholar 

  115. 115.

    Bey M, Berrin JG, Poidevin L, Sigoillot JC: Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes. Microb Cell Fact. 2011, 10: 113-

    Google Scholar 

  116. 116.

    Akbarzadeh A, Ranaei Siadat SO, Motallebi M, Zamani MR, Barshan Tashnizi M, Moshtaghi S: Characterization and high level expression of acidic endoglucanase in Pichia pastoris. Appl Biochem Biotechnol. 2014, 172: 2253-2265.

    Google Scholar 

  117. 117.

    Jager G, Girfoglio M, Dollo F, Rinaldi R, Bongard H, Commandeur U, Fischer R, Spiess AC, Buchs J: How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis. Biotechnol Biofuels. 2011, 4: 33-

    Google Scholar 

  118. 118.

    Tsai SL, Goyal G, Chen W: Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production. Appl Environ Microbiol. 2010, 76: 7514-7520.

    Google Scholar 

  119. 119.

    Kim S, Baek SH, Lee K, Hahn JS: Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and beta-glucosidase. Microb Cell Fact. 2013, 12: 14-

    Google Scholar 

  120. 120.

    Hyeon JE, Yu KO, Suh DJ, Suh YW, Lee SE, Lee J, Han SO: Production of minicellulosomes from Clostridium cellulovorans for the fermentation of cellulosic ethanol using engineered recombinant Saccharomyces cerevisiae. FEMS Microbiol Lett. 2010, 310: 39-47.

    Google Scholar 

  121. 121.

    Ilmen M, den Haan R, Brevnova E, McBride J, Wiswall E, Froehlich A, Koivula A, Voutilainen SP, Siika-Aho M, la Grange DC, Thorngren N, Ahlgren S, Mellon M, Deleault K, Rajgarhia V, van Zyl WH, Penttila M: High level secretion of cellobiohydrolases by Saccharomyces cerevisiae. Biotechnol Biofuels. 2011, 4: 30-

    Google Scholar 

  122. 122.

    Romanos MA, Scorer CA, Clare JJ: Foreign gene expression in yeast: a review. Yeast. 1992, 8: 423-488.

    Google Scholar 

  123. 123.

    Boer H, Teeri TT, Koivula A: Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotechnol Bioeng. 2000, 69: 486-494.

    Google Scholar 

  124. 124.

    Li YL, Li H, Li AN, Li DC: Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris. J Appl Microbiol. 2009, 106: 1867-1875.

    Google Scholar 

  125. 125.

    Sadie CJ, Rose SH, den Haan R, van Zyl WH: Co-expression of a cellobiose phosphorylase and lactose permease enables intracellular cellobiose utilisation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2011, 90: 1373-1380.

    Google Scholar 

  126. 126.

    Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A: Cocktail delta-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb Cell Fact. 2010, 9: 32-

    Google Scholar 

  127. 127.

    Khramtsov N, McDade L, Amerik A, Yu E, Divatia K, Tikhonov A, Minto M, Kabongo-Mubalamate G, Markovic Z, Ruiz-Martinez M, Henck S: Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass. Bioresour Technol. 2011, 102: 8310-8313.

    Google Scholar 

  128. 128.

    Olson DG, McBride JE, Shaw AJ, Lynd LR: Recent progress in consolidated bioprocessing. Curr Opin Biotechnol. 2012, 23: 396-405.

    Google Scholar 

  129. 129.

    Kricka W, Fitzpatrick J, Bond U: Metabolic engineering of yeasts by heterologous enzyme production for degradation of cellulose and hemicellulose from biomass: a perspective. Front Microbiol. 2014, 5: 174-

    Google Scholar 

  130. 130.

    Yamada R, Nakatani Y, Ogino C, Kondo A: Efficient direct ethanol production from cellulose by cellulase- and cellodextrin transporter-co-expressing Saccharomyces cerevisiae. AMB Express. 2013, 3: 34-

    Google Scholar 

  131. 131.

    Merico A, Capitanio D, Vigentini I, Ranzi BM, Compagno C: How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis. J Biotechnol. 2004, 109: 139-146.

    Google Scholar 

  132. 132.

    Liu B, Gong X, Chang S, Yang Y, Song M, Duan D, Wang L, Ma Q, Wu J: Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis. J Biotechnol. 2009, 143: 95-102.

    Google Scholar 

  133. 133.

    Aden A, Ruth M, Ibsen K, Jechura J, Neeves K, Sheehan J, Wallace B, Montague L, Slayton A, Lukas J: Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis for corn stover. In DTIC Document. NREL; 2002.

  134. 134.

    Ziegelhoffer T, Raasch JA, Austin-Phillips S: Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobacco. Mol Breeding. 2001, 8: 147-158.

    Google Scholar 

  135. 135.

    Dai Z, Hooker BS, Quesenberry RD, Thomas SR: Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification. Transgenic Res. 2005, 14: 627-643.

    Google Scholar 

  136. 136.

    Hood EE, Love R, Lane J, Bray J, Clough R, Pappu K, Drees C, Hood KR, Yoon S, Ahmad A: Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J. 2007, 5: 709-719.

    Google Scholar 

  137. 137.

    Oraby H, Venkatesh B, Dale B, Ahmad R, Ransom C, Oehmke J, Sticklen M: Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol. Trangenic Res. 2007, 16: 739-749.

    Google Scholar 

  138. 138.

    Mei C, Park SH, Sabzikar R, Ransom C, Qi C, Sticklen M: Green tissue specific production of a microbial endo cellulase in maize (Zea mays L.) endoplasmic reticulum and mitochondria converts cellulose into fermentable sugars. J Chem Technol Biotechnol. 2009, 84: 689-695.

    Google Scholar 

  139. 139.

    Mahadevan SA, Wi SG, Kim YO, Lee KH, Bae H-J: In planta differential targeting analysis of Thermotoga maritima Cel5A and CBM6-engineered Cel5A for autohydrolysis. Transgenic Res. 2011, 20: 877-886.

    Google Scholar 

  140. 140.

    Jung S, Lee DS, Kim YO, Joshi CP, Bae HJ: Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5' amplification promoting sequence. Plant Mol Biol. 2013, 83: 317-328.

    Google Scholar 

  141. 141.

    Caspi J, Barak Y, Haimovitz R, Gilary H, Irwin DC, Lamed R, Wilson DB, Bayer EA: Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A. Syst Synth Biol. 2010, 4: 193-201.

    Google Scholar 

  142. 142.

    Klose H, Röder J, Girfoglio M, Fischer R, Commandeur U: Hyperthermophilic endoglucanase for in planta lignocellulose conversion. Biotechnol Biofuels. 2012, 5: 63-

    Google Scholar 

  143. 143.

    Oey M, Lohse M, Kreikemeyer B, Bock R: Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 2009, 57: 436-445.

    Google Scholar 

  144. 144.

    Ziegelhoffer T, Raasch JA, Austin-Phillips S: Expression of Acidothermus cellulolyticus E1 endo-β-1,4-glucanase catalytic domain in transplastomic tobacco. Plant Biotechnol J. 2009, 7: 527-536.

    Google Scholar 

  145. 145.

    Yu L-X, Gray BN, Rutzke CJ, Walker LP, Wilson DB, Hanson MR: Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco. J Biotechnol. 2007, 131: 362-369.

    Google Scholar 

  146. 146.

    Gray B, Yang H, Ahner B, Hanson M: An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts. Plant Mol Biol. 2011, 76: 345-355.

    Google Scholar 

  147. 147.

    Gray BN, Ahner BA, Hanson MR: High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions. Biotechnol Bioeng. 2009, 102: 1045-1054.

    Google Scholar 

  148. 148.

    Petersen K, Bock R: High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome. Plant Mol Biol. 2011, 76: 311-321.

    Google Scholar 

  149. 149.

    Krichevsky A, Meyers B, Vainstein A, Maliga P, Citovsky V: Autoluminescent plants. PLoS One. 2010, 5: e15461-

    Google Scholar 

  150. 150.

    Bock R: Genetic engineering of the chloroplast: novel tools and new applications. Curr Opin Biotechnol. 2014, 26: 7-13.

    Google Scholar 

  151. 151.

    Ruf S, Karcher D, Bock R: Determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci U S A. 2007, 104: 6998-7002.

    Google Scholar 

  152. 152.

    Nuttall J, Vine N, Hadlington JL, Drake P, Frigerio L, Ma JK: ER-resident chaperone interactions with recombinant antibodies in transgenic plants. Eur J Biochem. 2002, 269: 6042-6051.

    Google Scholar 

  153. 153.

    Klose H, Günl M, Usadel B, Fischer R, Commandeur U: Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development. Biotechnol Biofuels. 2013, 6: 53-

    Google Scholar 

  154. 154.

    Hartati S, Sudarmonowati E, Park YW, Kaku T, Kaida R, Baba K, Hayashi T: Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon. Plant Physiol. 2008, 147: 552-561.

    Google Scholar 

  155. 155.

    Ransom C, Balan V, Biswas G, Dale B, Crockett E, Sticklen M: Heterologous Acidothermus cellulolyticus 1,4-beta-endoglucanase E1 produced within the corn biomass converts corn stover into glucose. Appl Biochem Biotechnol. 2007, 137-140: 207-219.

    Google Scholar 

  156. 156.

    Zhang Q, Zhang W, Lin C, Xu X, Shen Z: Expression of an Acidothermus cellulolyticus endoglucanase in transgenic rice seeds. Protein Expres Purif. 2012, 82: 279-283.

    Google Scholar 

  157. 157.

    Maloney VJ, Mansfield SD: Characterization and varied expression of a membrane-bound endo-beta-1,4-glucanase in hybrid poplar. Plant Biotechnol J. 2010, 8: 294-307.

    Google Scholar 

  158. 158.

    Syngenta. [], [http://www.syngenta.com/country/us/en/enogen/Pages/Home.asp]

  159. 159.

    Hood EE, Devaiah SP, Fake G, Egelkrout E, Teoh K, Requesens DV, Hayden C, Hood KR, Pappu KM, Carroll J, Howard JA: Manipulating corn germplasm to increase recombinant protein accumulation. Plant Biotechnol J. 2012, 10: 20-30.

    Google Scholar 

  160. 160.

    Harrison MD, Geijskes J, Coleman HD, Shand K, Kinkema M, Palupe A, Hassall R, Sainz M, Lloyd R, Miles S, Dale JL: Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane. Plant Biotechnol J. 2011, 9: 884-896.

    Google Scholar 

  161. 161.

    Harrison MD, Geijskes RJ, Lloyd R, Miles S, Palupe A, Sainz MB, Dale JL: Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane. Mol Biotechnol. 2014, 56: 795-802.

    Google Scholar 

  162. 162.

    Kaida R, Kaku T, Baba K, Oyadomari M, Watanabe T, Nishida K, Kanaya T, Shani Z, Shoseyov O, Hayashi T: Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood. Mol Plant. 2009, 2: 904-909.

    Google Scholar 

  163. 163.

    Taylor Ii LE, Dai Z, Decker SR, Brunecky R, Adney WS, Ding S-Y, Himmel ME: Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels. Trends Biotechnol. 2008, 26: 413-424.

    Google Scholar 

  164. 164.

    Sainz M: Commercial cellulosic ethanol: rhe role of plant-expressed enzymes. In Vitro Cell Dev-Pl. 2009, 45: 314-329.

    Google Scholar 

  165. 165.

    Brunecky R, Selig M, Vinzant T, Himmel M, Lee D, Blaylock M, Decker S: In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize. Biotechnol Biofuels. 2011, 4: 1-

    Google Scholar 

  166. 166.

    Zhang D, VanFossen A, Pagano R, Johnson J, Parker M, Pan S, Gray B, Hancock E, Hagen D, Lucero H, Shen B, Lessard P, Ely C, Moriarty M, Ekborg N, Bougri O, Samoylov V, Lazar G, Raab RM: Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes. Bioenerg Res. 2011, 4: 276-286.

    Google Scholar 

  167. 167.

    de O Buanafina MM, Fescemyer H: Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm. Planta. 2012, 236: 513-523.

    Google Scholar 

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Acknowledgements

This work was supported by the BMBF Forschungsinitiative `BioEnergie 2021 - Forschung für die Nutzung von Biomasse' (0315487A-C) and the Cluster of Excellence `Tailor-made Fuels from Biomass' (EXC 236), which is funded through the Excellence Initiative by the German federal and state governments to promote science and research at German universities.

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  1. Megan Garvey

    Present address: School of Medicine, Deakin University, CSIRO Australian Animal Health Laboratory, 5 Portarlington Rd, Newcomb, 3219, VIC, Australia

  2. Holger Klose

    Present address: Institute for Botany and Molecular Genetics, RWTH Aachen University, Worringerweg 3, Aachen, 52074, Germany

Affiliations

  1. Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany

    Camilla Lambertz, Megan Garvey, Johannes Klinger, Dirk Heesel, Holger Klose, Rainer Fischer & Ulrich Commandeur

  2. Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, Aachen, 52074, Germany

    Rainer Fischer

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  1. Camilla Lambertz
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Lambertz, C., Garvey, M., Klinger, J. et al. Challenges and advances in the heterologous expression of cellulolytic enzymes: a review. Biotechnol Biofuels 7, 135 (2014). https://doi.org/10.1186/s13068-014-0135-5

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Keywords

  • Cellulase
  • Heterologous expression
  • Cellulosome
  • Consolidated bioprocessing
  • Industrial processing

Biotechnology for Biofuels

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