To optimize improvements to deconstruction due to overexpression of lignocellulolytic enzymes in planta, researchers have sought to increase the percent of plant total soluble protein (TSP) that the enzymes represent and to decrease detrimental plant phenotypes caused by enzyme accumulation. In many studies, hydrolase levels remain relatively low (1-5% TSP) as do the % increases in saccharification. Figure 7 summarizes strategies being used to improve in planta expression of GHs. These include organelle targeting, codon optimization, promoter enhancement, and transient expression. Some of these methods as well as other approaches to regulate hydrolase activity, such as use of inteins, are also being tested for their ability to mitigate plant dwarfism or other physical defects that can accompany in planta GH expression.

Organelle targeting is a key factor in optimal accumulation of cell wall degrading enzymes in plants. Most previous studies have examined accumulation of enzymes in the apoplast, cytosol, and vacuole (Sticklen 2006; Taylor et al. 2008; Sainz 2009). Targeting the transgene to incorporate into the chloroplast genome has also been shown to induce accumulation of large amounts of foreign proteins (Oey et al. 2009). For example, a collection of GHs and related proteins from T. reesei expressed in the chloroplast had higher activity and pH and temperature stability compared with the same proteins expressed in E. coli (Verma et al. 2010). A crude cocktail derived from lines expressing each enzyme released up to ~3500% more glucose from biomass compared with a commercial enzyme cocktail, though it is not clear that the study was conducted with the same amount of protein in both samples (Verma et al. 2010). Another study showed that with chloroplast targeting, tobacco could accumulate four cell wall degrading enzymes at levels up to 40% TSP (Petersen et al. 2011). However, if selected for homoplasty, meaning all chloroplasts were transformed, this resulted

Figure 7. Strategies for improving glycosyl hydrolase expression in plants to enhance the quality of biomass for biochemical production of biofuels.

in pigment-deficient mutants unable to grow autotrophically. Even if heteroplastic, transformant had to be grown on a sucrose-rich medium. This is in contrast to the 70% TSP achieved without any severe phenotype when the tobacco expressed a protein antibiotic (Oey et al. 2009). The authors suggest that the chloroplastic GHs might sequester intermediates needed in plant metabolism. Based on these results, chloroplast transformation harbors the following promises: a significantly better biological enzyme factory than bacteria; an environment suitable for high accumulation of enzymes versus the apoplast, vacuole, cytosol, etc.; and a setting amendable for producing plants that could express a multitude of cell wall degrading enzymes. However, further strategies are needed to combat deleterious growth effects.

Codon optimization is a simple way to enhance heterologous expression levels. All organisms and DNA-containing organelles contain particular preferences for codon usage. Foreign genes may not adhere to these preferences and likely due to depletion of rare tRNAs these codons may diminish expression. Arabidopsis expressing codon optimized

D. thermophilum XynA and XynB resulted in a TSP of 14% and 3%, respectively, and a 14% improvement in xylose release compared with the wild-type plant (Borkhardt et al. 2010). The codon optimized expression of XynA and XynB in the apoplast allowed for significantly higher accumulation of these enzymes than what was seen in earlier un-optimized examinations with similar xylanases from S. olivaceoviridis (Yang et al. 2007).

Increasing promoter activity is another key target to improving TSP levels of heterologous GHs. The ideal promoters are those with strong location specific and/or inducible activity (Taylor et al. 2008). For instance, accumulation of 5.8% TSP was obtained by driving the gene encoding a thermostable fi-glucosidase, BglB from Thermotoga mamma, under the control of the rbcS-1A promoter and with targeting to the chloroplast (Jung et al. 2010). This is a light-regulated promoter that controls the transcription of Ribulose-1,5-bisphosphate carboxylase oxygenase small subunit. In another study, a TSP of 6.1% for the E1 cellulase under the control of the synthetic Mac promoter was achieved for apoplast targeting in rice (Chou et al. 2011). This was higher than the 4.9% TSP obtained in another study using the Cauliflower Mosaic Virus 35S promoter (Oraby et al. 2007).

Besides enhancing expression of heterologous hydrolases, other challenges associated with GH expression are developmental defects including stunted growth, severe pigment deficiency, enhanced disease susceptibility, poor seeds, and poor growth (Dai et al. 1999; Skjot et al. 2002; Harholt et al. 2010; Gray et al. 2011). Common strategies to avoid these deleterious effects are conceptually similar to those used to enhance expression and include selectively targeting hydrolase expression to storage organelles, use of inducible or developmentally regulated promoters, as well as employing thermophilic enzymes with low activity at ambient temperatures, as mentioned previously (Taylor et al. 2008; Jung et al. 2012). Inteins are another effective means to regulate in planta GH activity. An intein is a protein that can catalyze its own removal from and the subsequent rejoining of two flanking protein segments, i. e., the exteins (Sharma et al.

2006) . Inteins have been engineered to be activated by a variety of different external stimuli, including pH, temperature, and small molecules, providing good potential for use as a means to control activity of celluolytic enzymes in plants (Skretas et al. 2005; Sharma et al. 2006). Recently, a thermo-regulated intein was used to control the activity of a thermostable xylanase, XynB from Dictyoglomus thermophilum, expressed in maize (Shen et al. 2012). Without the intein the xylanase greatly reduced the plant seed mass and fertility, but these deleterious phenotypes were largely restored by the insertion of the intein. Xylanase activity was retained after the temperature was elevated to induce intein excision. Biomass from the xylanase-intien expressing plants released approximately 45% more sugar than that from the unmodified plants (Shen et al. 2012). Future directions might be to express a cocktail of hydrolases without any significant effects on phenotype.