Cellulase Genes In Wheat And Tobacco Apoplast Biology Essay

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Lignocellulose is basically considered as a renewable material and is the important component of many plants. The Lignocellulose mainly consists of three important components; they are cellulose, hemicellulose and lignin. In addition, traces of pectin can be found in the lignocellulosic waste material(Sánchez, 2008). The cell wall of the plant is made of basically made of lignocelluloses and protects against the invaders such as the insects and the microorganism.

Apart from agriculture, lignocellulosic wastes are produced in enormous amounts by various industries like forestry, paper and food industry (Kim and Dale, 2004). Cellulose is a complex organic biopolymer and represents itself to be one of the most abundant and also the most prevailing renewable waste material from agriculture (Bhat and Bhat, 1997).In natural conditions, the purest form of cellulose is rarely found and is most commonly found to be in a matrix of other biopolymers such as hemicellulose and lignin (Figure 1)(Chang, 2007; Sticklen, 2006). Biopolymers such as cellulose, hemicellulose and lignin are found to be bountiful and cellulose alone accounts for about 100 billion tons of the yearly biomass production. Overall, cellulose has been conceded as the most important unlimited sources of raw material for different value added products(Hartati et al., 2008).

Figure 1 The cell wall contains the cellulose microfibrils, hemicellulose, pectin, lignin and soluble proteins. The above figure is adopted from (Sticklen, 2006).

1.1.1 Important components of the plant cell wall Cellulose

The plant produces enormous amounts of cellulose every year. This enormously produced polysaccharide is considered as the biggest reservoir of organic carbon on earth(Festucci Buselli et al., 2007).The polysaccharide is about 15 to 30% of the dry mass from primary and makes up to 40% of the secondary cell walls. In the cell walls the polysaccharide is found in the form of 30 nm diameter microfibrils. These microfibrils are unbranched polymers with an estimated value of about 15,000 anhydrous glucose molecules organized in β‑1, 4 linkages (Chang, 2007; Zhao et al., 2007) (Figure 2). The microfibril consists of crystalline regions and is arranged in parallel to each other. Inside the microfibril parallels, the cellulose molecules are tightly packed. Apart from the anhydrous regions, cellulose also has soluble regions otherwise called amorphous regions. The molecule in this region in particular are less compact, but are staggered and leads to strengthening of the overall structure of the cellulose molecule (Zhao et al., 2007). Currently, the polysaccharide cellulose is being used for ethanol production in commercial scale, because it is the polysaccharide for which degrading enzymes are available.

Figure 2 Cellulose showing β, 1-4 linkages. The figure was adopted from (Chang, 2007). Hemicellulose

The cellulose molecules are surrounded by other polysaccharides like hemicellulose or xyloglucans (Figure 3). Both dicotyledonous and monocotyledonous ones consist mainly of xyloglucans. However, in some monocotyledons (cereals and other grasses), cell walls consists of glucuronoarabinoxylans. The percentage of hemicellulose in the plant cell wall is about 20- 40% of the plant cell. Hemicellulose can also be converted to glucose like that of cellulose by enzymatic hydrolysis for the production of bioethanol(Sticklen, 2008).

Figure 3 Hemicelluloses mainly showing the pentose sugar xylose. The figure was adopted from (Chang, 2007). Lignin

Lignin is considered as the major constituent of secondary cell walls. The total percentage of lignin content in plant dry matter is about 10 to 25%. Lignin is composed of aromatic compounds covalently attached to cellulose and xylose with phenyl and ester bonds (Boerjan et al., 2003)(Figure 4). To date, neither the chemistry of lignin with cell-wall polysaccharides nor the biosynthesis of lignin pathway is well understood35. The main role of lignin is to protect the plant from invaders such as pathogens and insects (Mosier et al., 2005).

Figure 4 Shows the heterogeneous polymer containing aromatic rings. The figure was adopted from (Chang, 2007).

1.2 Microbial cellulases

The cellulose polysaccharide has not been utilized to its greatest extent because of its rigid polymer structure and surface irregularities. It's impossible for large molecules like enzymes and small molecules like water to penetrate the rigid structure of cellulose (Fan et al., 1980). For efficient use of cellulose from the lignocellulosic waste, microbial biodegradation of cellulose is suggested to be one of the finest strategies to produce desired biofuel. Its also well known that the biopolymer is recalcitrant to microbial action, but suitable pretreatments of the lignocellulosic biomass has resulted in break down of the lignin structure and making it more accessible for the enzymes to increase the rate of cellulose biodegradation (Lynd et al., 2002).

Degradation of lignocellulosic biomass is by the action of several microbial enzymes and the most vital of which are the cellulases. Cellulases are produced by a number of microorganisms and it contains several different enzyme classifications. The main function of cellulase is to break down the β-1, 4-D glucan linkages of cellulose to produce cellobiose and glucose(Watanabe and Tokuda, 2010). Microbes that are cellulolytic are carbohydrate degraders and use carbohydrate as their source for their growth. They don't have the capacity to metabolise lipids or protein as energy sources for growth (Lynd et al., 2002).In general, most of the cellulase producing microorganism include bacteria, fungi and actinomycetes .

Though, several microorganisms are known to produce cellulases only few have been extensively studied and also have been known to produce large amounts cellulases. One of which is a fungi called as T. reesei, which converts various sources of cellulose to glucose and the other one is a bacteria called as A. cellulolyticus .These are the two important microorganisms that are commercially exploited for cellulase preparation. The production of cellulases in commercial scale has been tried in the form of solid or submerged culture and continuous flow process. The fermentation process has cellulose as one of the major sources of substrates for large scale production of cellulases. Cellulases are called inducible enzymes and naturally induced by cellulose and lactose. However, the large scale production of cellulases by both the inducers has been very expensive and genetic modifications of the microorganism producing cellulases to improve the production and activity of the cellulase has been achieved but commercial production requires further improvements in bioethanol production(Sukumaran et al., 2005).

1.2.1 Plant cell wall degrading enzymes Cellulases

The cellulase system produced by microorganisms consists of either secreted or cell bound based on their mode of action and structural properties. There are three major types of cellulases; they are 1-4-β-D endoglucanases (EC3.2.14), 1-4-β-D-glucan exoglucanses (EC and β-glucosidases (EC. (Seiboth et al., 1997).

The main function of endoglucanase is to cut the long polysaccharide chain randomly in the amorphous sites to form oligosaccharides. Exoglucanses cut the long polysaccharide from its reducing or non reducing ends to form cellobiose and β glucosidases break down the disaccharide such cellobiose to fermentable glucose (Gow and Wood, 1988; Moloney et al., 1985). These three forms of enzyme act synergistically together to degrade cellulose to monomeric sugars. The two major cellulases

There are two major cellulases produced by T. reesei and A. cellulolyticus that has been studied extensively and also has been used commercially to produce bioethanol. The two are cellobiohydrolase 1 (CBH1) of T. reesei and endoglucanase (E1) 1 from A. cellulolyticus. CBH1 was identified during the Second World War and E1 was isolated from yellow stone national park (Bhat and Bhat, 1997; Mohagheghi et al., 1986). E1 and CBH1 enzymes are preferred over other cellulases because E1 is thermostable (Topt = 81 °C) and the later has thermostability up to 55°C.The E1 acidophilic cellulase (pH 4.5-5.5) grows well on submerged culture to hydrolyze cellulose to produce disaccharides like cellobiose efficiently for further downstream process like fermentation. Arrangement of CBH1 in T. reesei

Small-angle X-ray scattering analysis was first used to identify the structure of CBH1 from T. reesei. (Abuja et al., 1988). The CBH 1 enzyme is arranged in the form of a tadpole like shape with an isotropic head and a long flexible tail (Figure 6). The determination of the three dimensional structure of the catalytic domain of CBH I of T. reesei was by X-ray crystallography upon proteolytic cleavage of the CBD .The catalytic domain is arranged in the form of a large single domain protein with two large antiparalle1 β sheets that stack towards each other to form a β -sandwich (Rabinovich et al., 2002). The main function of CBH1 within the microbial system is to cleave cellulose chains from the reducing end and making cellobiose units available to β- glucosidase to form monomeric sugars (Divne et al., 1994; Rouvinen et al., 1990) . Though, the CBH1 is very active in T. reesei, the real question is whether this enzyme is active in a heterologous system such as bacteria, yeast and plants.

Figure 5 CBH1 from T. reesei by small angle X-ray scattering analysis. A= CBD and B=linker region (Abuja et al., 1988) Arrangement of E1 from A.cellulolyticus

It has been found that X-ray crystallography of this enzyme has showed the catalytic domain consisting of an (α/β) 8 barrel fold where the protein loops in the structure. Also, the enzymes contains 16-26 residues each and form the walls of a catalytic crevice of about 9 Å wide, 30 Å long and 10 Å deep(Sakon et al., 1996). The major question is how far has E1 been expressed in a heterologous system.

The catalytic mechanism of E1 is a double-displacement mechanism put forth originally by Koshland in1953. It involves three basic steps,

Initial binding of the substrate to the enzyme.

Acid-catalysed attack of an enzymatic nucleophile upon the anomeric centre to form a glycosyl-enzyme intermediate (Sakon et al., 1996).

The intermediate is hydrolysed by the base-catalysed attack of water upon the anomeric centre forming the product and returning the enzyme to its original protonation state (Sakon et al., 1996). Hemicellulase

Cellulose is trapped inside the hemicellulose and it has to be removed for the cellulases to access cellulose. The main constituent of hemicellulose is the β‑1, 4-xylan. To remove the xylan, xylanase are used that have both endo-and exo-activity (Warren, 1996). Ligninases

Degradation of lignin by microorganisms is poorly understood. The enzyme from Phanerochaete chrysosporium and Trametes versicolour are thought to be the most efficient white rot fungi to produce enzymes for lignin degradation (D'Souza et al., 1999). The three important families of Ligninases that are produced by fungi are laccases, manganese-dependent peroxidases and lignin peroxidises (Kirk and Farrell, 1987). Microbial fermentation of cellulases

There are basically two types of fermentation technology used for large scale production of cellulases. Submerged fermentation technology is one of the main technology that has extensively been reported for microbial production of cellulases. Cellulase production from T. reesei is a classic example of submerged fermentation technology (Mandels and Reese, 1957). The major carbon source in commercial production of cellulases is lignocellulosic biomass that includes pulses, rice, bagasse and various other agricultural residues (Ayub, 2002; Belghith et al., 2001; Romero et al., 1999; Wen et al., 2005). Until now, cellulases have been produced in batch process and currently attempts are being made in continuous and fed batch culture to avoid repression caused by large amounts of reducing sugar (Strauss et al., 1995).

The other rapidly growing technology for cellulase production is the solid state fermentation. It has gained interest solely because of the cost-effectiveness in producing cellulases. Besides the cost, this technology efficiently uses lignocellulosic biomass as a source of cellulase production. It is also been indicated that cellulase production through solid state fermentation has a ten-fold reduction in over all production cost. Though this technology is proving to be cost effective, large scale production of cellulases are produced by submerged fermentation technology(Sukumaran et al., 2005). The need for cellulases

Initially, industries such as the textile, leather, animal feed, food, detergent and the paper industry used toxic chemicals for many years to get their desired products. Usage of such toxic chemicals has resulted in contamination of soil, water and others leading to global environmental pollution. The use of microorganisms or enzymes has been very effective in the reduction of contamination caused by toxicants(Alcalde et al., 2006).Enzymes produced by microbial sources have a greater advantage over toxic chemicals as they make better use of raw materials, they save water and energy. In addition, the enzymes produced by the fungi and bacteria protect the forest natural cycle by breaking down deadwood into soil and replenishing the fertility of the forest soil(Turner et al., 2007). Hence enzymes produced by the microorganisms have been useful to mankind in the above mentioned ways.

Cellulase is one such enzyme that is known for not only bioconversion of biomasses, but also for paving the way to research in various industrial applications such as textile, animal feed, detergent and paper industry. Apart from the above mentioned applications, cellulases are currently being employed to convert lignocellulosic waste for for biofuel production (Turner et al., 2007).

1.3 Heterologous cellulase expression

Heterologous expression systems is also known as a powerful technique to produce large amounts of enzymes with increased activity(Dashtban et al., 2009). There are some eukaryotic organism whose sequences are found to be well conserved (Botstein and Fink, 1988) and also some of their molecular mechanisms are known to be similar. Molecular mechanisms such as intracellular transport, compartmentation and gene regulations such as cell cycle control (Nurse, 1990), signal transduction(Assmann, 1993; Holzenburg et al., 1993), chromatic structure (Sukumaran et al.) and vesicular trafficking along the secretory pathways(Bednarek and Raikhel, 1992) .

The need for heterologous expression was required because the cost involved producing cellulases from fungi was too high and also cellulose was not being converted efficiently to fermentable by native cellulolytic microorganisms due to product inhibition. Hence, heterologous expression of cellulases in non cellulolytic microorganisms was tried in non cellulolytic hosts because they were know to exhibit excellent product formation properties and produce functional heterologous expression of a cellulase system. In order to produce

1.3.1 Heterologous expression of endoglucanase1 (E1) and CBH1 from A. cellulolyticus and T. reesei The need for heterologous expression of E1 in E. coli and yeast

Although cellulose is an abundant biopolymer, cellulose is resistant to depolymerization. Cellulose becomes the source for ethanol production after it has been hydrolyzed to glucose. There is one method by which cellulose containing biomass can be converted to glucose, which is by using cellulase enzymes. Cellulase enzymes are found in several fungi and bacteria. However, cellulase produced by fungi especially from T.reesei is in large quantities and are preferred for ethanol production from cellulosic biomass(Lastick Deceased., 1996).

The only drawback in using T.reesei is that it's costly to produce ethanol using this fungus. This is because the fungus grows slowly and requires sugar for its growth and induction of enzymes. In addition cellulase produced by T.reesei gets inhibited by cellobiose and by glucose by the process of end product inhibition. Though the end product inhibition can be avoided by simultaneous saccharification and fermentation process (SSF), the SSF technology is expensive (Shoemaker et al., 1981). Since the cost of ethanol production is very high by using cellulases from T.reesei, alternative cellulase producing fungi and bacteria were studied and none of the cellulases producing bacteria could produce cellulases at levels produced by T.reesei.

To avoid the cost involved in bioethanol production by using fungal cellulases. A highly thermostable endoglucanase 1 isolated from A. cellulolyticus was expressed in E. coli and its thermo stability was found to be stable than that of the native E1. The host E. coli was chosen because it grows faster than fungus and also can be induced for the overproduction of the desired cellulase by chemicals such as IPTG. In addition, the endoglucanase produced by A. cellulolyticus is less inhibited by cellobiose than that of T.reesei and also the enzyme is active over a broad pH range, pH range at which yeast can convert glucose to ethanol(Lastick Deceased., 1996). Expression of E1 in Pichia pastoris (P. pastoris) and Streptomyces lividians (S. lividians)

However, E1 is expressed in E. coli in a truncated form because of indigenous proteolytic activity and is comparably active to that of the full length enzyme. This comparison was assayed in the presence of T. reesi cellobiohydrolase 1(CBH1). To avoid the truncated forms of E1 cellulase, the full length E1 was expressed in S. lividians and yeast, namely Pichia pastoris. The Pichia pastoris (P. pastoris) has been known as a useful host in heterologous expression of the proteins. The E1 cellulase produced by P. pastoris was heavily glycosylated but the enzyme activity was retained when compared to the native cellulase. In addition, the amount of E1 full length cellulase produced by S. lividians was less in comparison to the amount produced in yeast. Heterologous expression of E1 in E. coli was produced in large quantities and E. coli was considered to be the best host for the production of active endoglucanases. This is because the truncated form of the E1 enzyme is thermostable at 81 °C and the enzyme can be overproduced by using lac promoter for overproduction of the E1 cellulase when compared to yeast and S. lividians. The promoters used in yeast and S. lividians cannot be induced for overproduction of the E1 enzyme but can be used to produce required amount of heterologous protein. The truncated form of E1 contains the catalytic domain and it was found that the specific activity of the truncated form of the E1 enzyme was 10 °C higher than the full length enzyme(Adney, 1998).

Though the full length and the catalytic domain of E1 cellulase is expressed in bacteria, fungi and yeast at high levels the cost is associated with the type of substrate and pretreatment process being used to produce bioethanol. The look for cheaper hosts, substrates and the type of pretreatment is currently under investigation. Heterologous expression of cellobiohydrolase 1(CBH1) from T. reesi.

Microorganisms are currently not available for consolidated bioprocessing. Consolidated bioprocessing compared to other fermentation processes gives very large cost reductions(Dashtban et al., 2009). Therefore the need for suitable non-cellulolytic organisms exhibiting high product yields is being considered for heterologous cellulase production. Saccharomyces cerevisiae is considered to be a suitable host for heterologous expression of cellulases. The reason for S. cerevisiae being the attractive host is because it exhibits tolerance towards inhibitors found in hydrolyzates found after the pretreatment processes. The S. cerevisiae also coexpressed endoglucanase and β-glucosidase on cellulosic substrates (Den Haan et al., 2007). High level expression of CBH1, in particular, has been a great challenge for many years. As a result, high amounts of CBH1 enzyme was produced in S. cerevisiae from T. reesi but the activity of CBH1 was very low. This was basically due to hyperglycosilation of CBH1 produced by the system. Hence CBH1 was expressed in S. cerevisiae but the activity is significantly lower than the reported value (Penttilä et al., 1988; Reinikainen et al., 1992).Recombinant CBH1 has been expressed in other microbial systems but the activity of CBH1 is lower when compared to the native form of the enzyme produced by T. reesi. Suitable hosts are being investigated to avoid problems associated with glycosylation.

1.4 Cellulase improvement

1.4.1 Cellulase engineering

To date there are many cellulase enzymes that are well characterised, but in particular there is no enzyme suitable for the hydrolysis of cellulose at the industrial. However, these enzymes can be used as initiators for creating improved forms of cellulases in enhancing the economic value of the biofuel. To improve the enzymatic action of cellulase on cellulose the use of site directed mutagenesis has been extensively studied. The use of site directed mutagenesis is to improve the catalytic activity of the cellulases enzymes. Also, the use of protein engineering technology has been concentrated towards catalytic function and the role of various amino acids inside the catalytic domains of various cellulases(Maki et al., 2009). For example the active site of E1 from A. cellulolyticus was modified and was found that the process of saccharification in the pretreated yellow poplar increased by 12% when compared to the native E1 and E1 from A. cellulolyticus was found to be less inhibited in the presences of cellobiose (Baker et al., 2005; Himmel et al., 1999b).This indicates that the strategy used to engineer the catalytic site may serve to be an excellent method to increase the catalytic activity of cellulase enzymes.

To convert the lignocellulosic biomass more efficiently, Site directed mutagenesis was thought be used to engineer cellulase enzymes to increase their thermostability and pH optima when compared to their native counterparts (Schülein, 2000).There are basically two methods for the improvement of a cellulase and they are rational design and directed evolution.

1.4.2 Rational design

Rational design is based on the basis of selecting a suitable enzyme, amino acid sites that which require changes and characterization of the mutants. However, to use rational design knowledge of the protein structure is very important because to modify the amino acids structure of the protein plays an important role in understanding the enzyme activity(Percival Zhang et al.). Though there are well characterised cellulases, there is lack of knowledge about cellulase cellulose interactions and the synergistic behaviour within the cellulase components. The above mentioned factors are road blocks to improve cellulases by rational designing(Maki et al., 2009).

1.4.3 Directed evolution

To improve the enzymatic activity, stability and solubility, directed evolution is a new method that results in generation of mutants by the process of natural selection. These mutants are then screened by high-throughput screening methods. In comparison to the rational design directed evolution method is independent and doesn't require enzyme structure and synergistic behaviours of the enzyme(Maki et al., 2009). By directed evolution the changes in the protein structure can be studied (Himmel et al., 1999b; Johannes and Zhao, 2006). The directed evolution is a developing technology that utilises a variety of techniques including error-prone polymerase chain reaction (PCR), gene shuffling, site-saturation mutagenesis, and staggered extension process technology (Himmel et al., 1999a). Unfortunately, there has been limited success to date with this approach with most improvements being associated with thermal tolerance of the enzymes (Zhang et al., 2006)

1.4.4 The cellulose binding domain and catalytic activity

The cellulose binding domain and the catalytic binding in the hydrolysis of crystalline cellulose is a poorly understood concept. Previous studies have shown that the presence of cellulose binding domain and the catalytic domain has shown variation in the activity of the enzyme with and without cellulose binding domain. For example in the case of CBH1 from T. reesei, it was found that the binding of the CBH1 to the cellulose was reduced without its linker and cellulose binding domain (Jeoh et al., 2008). However, it was observed that the pattern of hydrolysis of crystalline cellulose is different from the hydrolysis of soluble forms of cellulose and what forms must be of cellulose has been used in a particular study. In addition to the activity differences, there are reports of cellulose binding domains capable of breaking down cellulose fibres in the absence of cellulase enzyme .There are other studies in which addition of cellulose binding domains to the complete enzyme has no change in the hydrolysis of cellulose and also proves that cellulose binding domain must be attached to the catalytic domain of the cellulase enzyme (Carrard et al., 2000; Din et al., 1991).Contrary to the above statement literature shows by adding free cellulose binding domains, in the presence of complete cellulase enzyme will release more amount of sugars from crystalline cellulose. This particular study suggests that by adding free cellulose binding domains to the completes enzyme may play some important role in the breakdown of the crystalline cellulose. However, this hypothesis was tested by adding cellulose binding domain in the absence of the complete enzyme and was found that there was no sugar released during this process (Lemos et al., 2003).The function of cellulose binding domain is not very clear in the literature and further research using x-ray crystallographic studies can help to improve the current knowledge of interactions between the cellulose binding domain and the cellulose substrate. It is possible that variation in the activity of the cellulase enzymes may depend upon the presence and absence of cellulose binding domain. For example in the case of endoglucanase 1 from A. cellulolyticus the catalytic domain is highly thermostable than the native E1 cellulase and it could also be possible that cellulase derived from different organisms have varying roles in their enzymatic function.

1.5 Heterologous expression of cellulases in planta

The expression of cellulases within the plant system is considered to be a short term strategy to improve bioethanol production. This is because the worldwide oil reserves are getting scarcer and the demand for energy is increasing at a very high level. Hence the need for sustainable and cost effective technology is required to meet the increasing demands(Taylor II et al., 2008).

1.5.1 Plant genetic engineering

Genetic engineering of plants is a powerful tool used to study gene expression in plants. Plant transformation is also known for its contribution to understand the gene regulation and plant development. Plant transformation studies have allowed the study of gene manipulation and biochemical process that cannot be studied or manipulated by conventional breeding methods. Plant genetic engineering has lead to many value based agricultural crops that have been proved to potentially increase food security in developing countries and produce nutriceutical products that benefit human health worldwide(Potenza et al., 2009). Though there are many benefits achieved from transgenic plants there were always questions related to the safety of the transgenic plants to the environment and the consumers. This issue was taken seriously and efforts were made to avoid the adverse effects of the genetically engineered plants by controlling the gene expression by expressing the genes to specific parts of the plants temporally and spatially. The answer to this is the promoters that drive the transgene for controlled expression. Introduction to promoters

Promoters are DNA sequences found upstream of the gene of interest that get recognised by specific protein responsible for the transcription to occur (Hill, 2001). The transcription initiation starts with the initial interaction of RNA polymerase II to the sequence elements of the promoter and the binding brings all the transcription factors that helps in the expression of the transgene. The promoters are very important in controlling overall expression of the gene by initiating or suppressing transcription at appropriate times and locations. Types of plant promoters

The promoters that are used in plant expression are of different types and are very specific in controlling transgene expression. There are viral, plant constitutive promoters, tissue specific promoters, inducible promoters and synthetic promoters (Dale et al., 2002). Constitutive promoters of viral and plant origin for heterologous expression of cellulases

Common promoters used for the over expression of the transgenes were obtained from different plants infected with different virus, one such promoter was cauliflower mosaic virus (CaMV) 35S promoter. 35S promoter was used mainly because it uses host nuclear RNA polymerases and basically they don't require trans - acting viral gene products for its function (Odell et al., 1985). This promoter like the other viral based promoters was obtained from double-stranded DNA viral genomes. This particular promoter is used quite often for transgenic expression in plants because it gives high level expression in all the regions of the transgenic plant. In particular, the CaMV 35S promoter has been used extensively used in expressing cellulases genes in both dicots and monocots because of its high level expression of the transgenes (Biswas et al., 2006; Jin et al., 2003b).

High level transgene expression was achieved by CaMV 35S and this lead to the derivation of several other viral based promoters such as the cassava vein mosaic virus (CsVMV) promoter, Australian banana streak virus (BSV) promoters , mirabilis mosaic virus (MMV) promoter and figwort mosaic virus (FMV) promoter .

Plant derived promoters such as ubiquitin do have the same function as that of CaMV 35S and such constitutive promoters when used to produce recombinant protein in seeds were found to be expressing recombinant proteins in leaves, pollen and roots of the plants. Recombinant protein could be exposed to pollinating insects and microorganism present surrounding the root system and that's the reason why constitutive promoters though express high level of recombinant protein in seeds were not found to be suitable for issues related to human health (Commandeur et al., 2003; Mae-Wan Ho, 1999). Due to high systemic expression of the recombinant proteins in the plants, tissue specific promoters were expected to limit recombinant protein expression before or after post harvest (Zuo and Chua, 2000). Green tissue specific promoters for heterologous expression of cellulases

The leaf supports the expression of genes that are well characterised and inducible when exposed to light. One of the best examples of light inducible genes are that genes that belong to the small subunit 1, 5-bisphosphate carboxylase (rbcS) multigene family. The rbcS promoters contain regulatory elements that are able to do tissue specific expression in the transgenic plants (Gilmartin and Chua, 1990). Although the light inducible promoter isolated from dicots and monocots have different conserved cis acting elements (Schaffner and Sheen, 1991) cellulase expression has been achieved using the rbcS promoter and was able to get high level cellulase expression in transgenic dicots and monocots (Dai et al., 2000b; Mei et al., 2009). The use of rbcS promoter limited the use of constitutive promoters for systemic expression and the look out for targeted expression of cellulases was achieved.

To date, E1 and CBH1 from A. cellulolyticus and T.reesei are the two main important cellulases that have been studied extensively by heterologous expression in various plants or crops such as alfalfa, Arabidopsis, maize, rice, tobacco and barley (Taylor II et al., 2008; Taylor et al., 2008). In addition to this high expression levels have been achieved in plants via, transcriptional post transcriptional and post translational modifications. Literature also indicates that the expression of E1 and CBH1 is based on the targeting organelle. Studies have also indicated that the expression of E1 catalytic domain and not the native E1 could be accumulated at high levels because of instability of the native E1.problems related to protein instability may be avoided if the factors mentioned above are considered. Sub-cellular targeting

Sub-cellular targeting was used as common method to get high level expression of recombinant proteins because the accumulation of the recombinant protein in designated compartment was known for its processes of proper folding, assembly, glycosylation and increased stability, as compared to the cytosol (Horn et al., 2004; Sticklen, 2006). When the cellulase enzyme was targeted to various compartments for example endoplasmic reticulum, the recombinant enzyme was expected to fold properly because of the presence of molecular chaperons which keep the cellulase enzyme away from the cytoplasmic metabolic activities, to avoid the degradation of the enzyme by proteases. All of these contributed to protein stability and, hence, sub-cellular targeting was considered to be helpful in determining the final yield of the recombinant proteins(Schillberg et al., 2002). In addition, the advantage of targeting heterologous enzymes such as cellulases (in this study) was that the enzyme could be extracted from the fresh or dry transgenic biomass which was considered as a part of total soluble protein (TSP), which can be added directly to the pretreated plant biomass for the break down of cellulose to fermentable sugars (Andersson-Gunnerås et al., 2006; Salehi et al., 2005).

There are many glycosyl hydrolases expressed in different compartments of the plant and the amount of protein accumulation also varied based on the choice of target. So far, on an average apoplast has been the considered as the best target location for the expression of stable cellulases. The highest amount of cellulase recorded in literature was that of endoglucanase 1 from A. cellulolyticus in Arabidopsis (Table 1).Table 1 also indicates the percentage of cellulase accumulation in different compartments of the plants in the literature.