Switchgrass is considered a prime candidate as a second generation biofuel feedstock because it can produce more ethanol per unit area and triple the net energy content than ethanol derived from corn grains (Bouton 2007). However, current estimates show that it requires 45 percent more fossil fuel energy to yield one liter of ethanol from two and a half kg of switchgrass feedstock than the energy in that one liter of ethanol fuel produced (Pimentel and Patzek 2005). The average cost to produce a liter of ethanol from switchgrass feedstock is approximately 54 cents, which is nearly nine cents higher than that for corn grains (Pimentel and Patzek 2005). One of the major cost factors in converting switchgrass feedstock into bioethanol is that of microbial enzymes, which are used to hydrolyze and break down the lignocellulosic biomass into fermentable sugars that can be used for biofuel production (Ragauskas et al. 2006). Presently, microbial hydrolysis enzymes are manufactured in large industrial bioreactors (Lynd et al. 2008). This process is extremely expensive and consequently, the cost of enzymes to produce one gallon of ethanol from lignocellulosic feedstock is roughly 30 cents per gallon (Bothast and Schlicher 2005).

In order to combat the high cost of microbial hydrolysis enzymes, current investigations are working towards expressing cell-wall degrading enzymes in important crop species. The most well studied cell-wall degrading enzymes are the cellulases, a family of enzymes that are naturally found in fungi, bacteria, and some animals (Sukumaran et al. 2005). These enzymes hydrolyze cellulose to produce glucose, cellobiose, and cellooligosaccharides (Sukumaran et al. 2005). There are three major types of cellulase enzymes: cellobiohydrolases, endo-1,4-p-glucanases, and P-glucosidases (Sukumaran et al. 2005). All three types of cellulase enzymes work collectively to break down cellulose into glucose monomer subunits that can then be fermented to yield bioethanol.

The best-studied of the cellulase enzymes is endo-1,4-p-glucanase. In 2000, Ziegler et al. inserted the catalytic domain of the endo-1,4-p-D- glucanase E1 gene (subsequently referred to as E1) from Acidothermus cellulolyticus into Arabidopsis and targeted protein localization to the apoplast (Ziegler et al. 2000). The authors were able to obtain levels of recombinant endoglucanase E1 between 0.01 to 25.7 percent of the total soluble protein (TSP). Novel zymogram assays further confirmed that the catalytic endoglucanase domain was biologically active (Ziegler et al.

2000). A similar study was performed in transgenic potato in which the entire endoglucanase E1 gene from A. cellulolyticus was targeted to mature leaves. Full-length recombinant endoglucanase E1 protein accounted for 2.6 percent of TSP in these transgenic potato plants (Dai et al. 2000a), which is an improvement over the 1.3 percent of partial endoglucanase E1 in TSP extracts of tobacco plants that were transformed using the same method (Dai et al. 2000b).

The successful expression and production of cell wall degrading enzymes in model plant species, such as Arabidopsis and tobacco, opened the door for utilizing this strategy in bioenergy crops. In 2007, Oraby et al. inserted the catalytic domain of the endoglucanase E1 gene from A. cellulolyticus into the nuclear genome of embroygenic rice calli via Agrobacterium transformation (Oraby et al. 2007). After regenerating transgenic plants, the E1 enzyme accounted for 2.4 to 4.9 percent of TSP in rice leaves. The presence of E1 also greatly enhanced the conversion of cellulose to glucose in pre-treated transgenic rice straw (Oraby et al. 2007). That same year, Ransom et al. inserted the same partial endoglucanase E1 gene, containing the catalytic domain, into corn embryogenic calluses (Ransom et al. 2007). The construct was placed under control of the cauliflower mosaic virus 35S promoter and introduced via particle bombardment (Ransom et al. 2007). Using this method, the authors were able to obtain up to 1.16 percent of biologically active recombinant endoglucanase E1 in TSP extracts (Ransom et al. 2007).

A recently published study performed by researchers from Agrivida Inc. (Medford, MA) investigated expressing two xylanase genes in maize under the direction of two different promoters (Gray et al. 2011). Xylanases are another family of cell wall degrading enzymes that act in correlation with cellulases to convert hemicellulose and cellulose into fermentable pentose sugars. The xynB gene from Clostridium stercorarium and the bsx gene from Bacillus sp. were cloned and optimized for expression in maize. After removing bacterial secretion signals, each gene was fused to two signal peptides individually: the barley a-amylase signal peptide sequence (BAASS), which targets protein accumulation to the cell wall, or the rice glutelin B-4 signal peptide (GluB4SP), which would allow for kernel-specific expression. The xylanase sequences that were fused to BAASS were placed under control of the constitutive rice rubi3 promoter, whereas the sequences that were fused to GluB4SP were directed by the rice GluB-4 gene promoter. All constructs were inserted into embryogenic calluses by Agrobacterium — mediated transformation. After transformed plants were regenerated, all of the T0 transgenic maize plants that constitutively expressed both xylanase genes displayed severely stunted growth phenotypes. In GluB4SP transgenic plants, where xylanase expression was directed to the seeds, the plants exhibited normal somatic tissue development, however, the corn grains appeared shriveled. Constitutive expression of both xylanase genes resulted in relatively low accumulation of BSX and XYB proteins in corn stover (0.1 percent TSP). Given that transgenic plants were undersized, higher levels of BSX and XYB accumulation may be lethal to the plant. However, seed specific expression of BSX and XYB resulted in up to four and 16.4 percent TSP, respectively. Presently, further research is being conducted to control xylanase activity and expression in an effort to prohibit negative growth phenotypes associated with expression of these genes in maize. In another case, a gene encoding a thermostable GH10 xylanase, Xy110B, from the hyperthermophilic bacterium Thermotoga maritima, was expressed in transplastomic tobacco plants (Kim et al. 2011). The accumulation levels of the enzymatically active Xy110B were between 11 and 15 percent of the total soluble protein in tobacco leaves. The enzyme displayed "exceptional" thermostability and catalytic activities over methylglucuronoxylose (MeGXn), a major form of xylan in woody plants. The enzyme was also biologically active, hydrolyzing MeGXn into fermentable sugars between 40 and 90°C, and was stable in dry and stored leaves. The transplastomic plants, as well as the progenies, appeared morphologically normal. Due to the harsh pretreatments needed for lignocellulosic feedstocks, selection of thermostable and extreme pH tolerant cellulases and xylanases is quite important for the recombinant enzymes to remain active after the pretreatments. Alternatively, one can work with engineers to develop milder pretreatment conditions and choose appropriate enzymes that can survive the best for those conditions. Moreover, the possibility to bypass pretreatment in certain transgenic alfalfa plants has been reported (Chen and Dixon 2007).

A similar strategy could be used to improve switchgrass as a feedstock. Cellulase enzymes need to be added to the switchgrass feedstock during alcohol production in order to hydrolyze cellulose and produce sugars for fermentation. Cellulases normally include endoglucanase, exoglucanase, and cellobiase (Keshwani and Cheng 2009) and the cost of added cellulases to the process is one of the remaining major economical obstacles for commercial alcohol production from lignocellulosic feedstocks. Currently, no reports have investigated expressing cellulase genes in switchgrass, a strategy that would no doubt facilitate saccharification and reduce the production cost.

Harsh pH and high temperature conditions during pretreatment of the feedstock is a major concern for the survival of the introduced enzyme(s). To overcome this problem, the mildest pretreatment, ammonia fiber explosion (AFEX), was applied to E1-transgenic tobacco biomass and roughly one third of the heterologous enzyme activity was retained. Alternatively, to circumvent the pretreatment stage, crude extract of the E1-transgenic rice plants was added to pretreated rice straw or corn stover and approximately 30 and 22 percent of the cellulose in these plants was converted into glucose, respectively (Sticklen 2006). The expression of cellulase genes in these plants did not have an obvious detrimental effect on plant growth and development. Targeting of these genes to cellular compartments could facilitate accumulation of the heterologous enzyme(s). In switchgrass, about 26 percent of the dry weight is hemicellulose (Keshwani and Cheng 2009), which is currently underutilized for fermentable sugar production and has a great potential for biofuel production in the future.

DOE-USDA awarded Agrivida Inc. (Medford, MA) a grant for producing switchgrass with cell wall degrading enzymes that would remain inactive during plant growth but become activated after harvest. Other laboratories are working to create transgenic switchgrass plants expressing endoglucanase (data unpublished). Using the information obtained from previous research in cereal crops (Oraby et al. 2007; Gray et al. 2011), combined with an efficient transformation system (Li and Qu 2011), switchgrass is a promising candidate for producing cell-wall degrading enzymes as a value-added trait. Introducing value-added traits, such as bioplastics and cell wall degrading enzymes, into important bioenergy crops will ultimately combat the high costs associated with turning the lignocellulosic feedstock into biofuels.