# Green Production and Biotechnological Applications of Cell Wall Lytic Enzymes
## 4 Production of CWLEs in Microbial and Plant Expression Systems
### Heterologous Expression of CWLEs in Plants
Delivery of recombinant proteins to lytic vacuoles (LVs) has already been attempted in the past, although this compartment was expected to be hydrolytic. Certain CWLEs such as cellobiohydrolase and endoglucanase accumulate at high yields in LVs [110]. However, vacuole sorting of CWLEs was strictly dependent on the development of LVs in different plant tissues, and protein accumulation was strongly reduced in both young and senescent leaves [111]. Interestingly, compartmentalized expression of CWLEs in crop plants concerns a very limited number of cellulases [102; 104; 105; 112; 113; 114]. [PERSON] and colleagues [115] succeeded in the apoplast accumulation of a truncated version of cellulase by expressing only the GH domain and excluding the CBM. Stable plant expression of CWLEs such as xylanases and \(\beta\)-glucosidasees were easier to obtain than that of cellulases, hinting that expression of cellulolytic enzymes _in planta_ has intrinsic limitations. In accordance with this observation, it has been shown that cellulase activity generates fragments, such as cellodext and cellobiose, which act as powerful DAMPs capable of inducing plant defense responses [91; 96]. Therefore, an uncontrolled activity of cellulolytic enzymes could lead to hyperactivation of immune responses and, consequently, largely affect plant growth and development. This scenario is avoided naturally by plants, since they are endowed with an enzymatic system that can inactivate, through specific modifications, the elicitor activity of different types of oligosaccharides [87; 88]. This plant characteristic should be further investigated and exploited to optimize CWLE production in this host. Inducible gene expression of CWLEs was achieved by using synthetic promoters such as those induced by chemical compounds (e.g., ethanol [116] and \(\beta\)-estradiol (i.e., XVE-based expression vector [117])) or by using endogenous promoters such as those induced by pathogens [118] and senescence [119] (Figure 3b). The strategy of using endogenous promoters is characterized by two major disadvantages: (i) the leakiness of the promoter and (ii) the developmental stage (e.g., senescence) at which the promoter is more active that and strongly reduce the expression potential. On the other hand, the use of synthetic promoters such as those induced by ethanol and \(\beta\)-estradiol makes the inducible systems not viable for field applications. In the last instance, an inducible activity of CWLEs can be achieved by expressing CWLEs with pH- and temperature-dependent activities such as those enzymes from acidophilic/halophilic and hyper-thermophilic bacteria (HCWLEs), respectively (Figure 3c). Plant expression of CWLEs with inducible activity (i.e., optimal activity at pH and temperature values extremely different from those of the plant) can be used to further reduce (dangerous) residual activities in vivo, thus preserving plant health and productivity. Notably, accumulation of HCWLEs was achieved in the apoplast of transgenic _Arabidopsis thaliana_ plants without adverse effects on plant growth [120,121]. When compared to their mesophilic enzymatic counterparts, HCWLEs have several advantages, which are mainly dependent on the temperature at which HCWLEs are active [122]. At industrial levels, high temperatures promote the relaxation of cell wall structures, thus facilitating CWLE substrate binding and preserving the  
Figure 3: Plant production of microbial CWLEs. Production of CWLEs as obtained by (**a**) constitutive expression and compartmentalization or (**b**) inducible gene expression. Advantages and disadvantages of each strategy are indicated in black and red, respectively. In (**a**), the represented CWLEs were successfully produced upon compartmentalized expression. (**c**) Theoretical optimal temperature of XynA from the mesophilic fungus _R. solani_, from the thermophilic bacterium _B halodurans_, and from the hyperthermophilic bacterium _T. martiima_, here reported as a representative set of CWL isoenzymes. Activity of XynA from _T.