# 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  
Plant expression of microbial CWLEs is a major challenge that biotechnologists are currently facing. Plants are desirable expression hosts since they are characterized by low production costs and high productivity. Moreover, they consume atmospheric CO2 through photosynthesis, which positively impacts global warming and points to the plant expression system as a valuable green alternative. The _in muro_ targeting of CWLEs may enhance the hydrolysis of cell wall polysaccharides, allowing an efficient conversion of fermentable sugars into biofuel-related compounds [82]. However, expression of CWLEs using plants as a heterologous system may impart unwanted and undesired side effects. CWLEs are produced by microbial pathogens to open a breach in the cell wall, concomitantly supporting the infection process [83]. Moreover, the degradation of cell wall polysaccharides provides sugars for sustaining the heterotrophic growth of phytopathogens inside the plant tissue [84]. In order to counteract the various CWLEs secreted by the pathogen, plants synthesize different specialized defense proteins that hinder their hydrolytic activity [85; 86; 87; 88]. Moreover, plants have evolved a complex system of cell surface receptors that promptly perceive CWLEs by sensing the products of their activity (i.e., cell wall derived fragments, formerly known as damage-associated molecular patterns (DAMPs) [89; 90; 91]), or to perceive the CWLEs themselves [92; 93] as microbe-associated molecular patterns (MAMPs) by specific recognition mechanisms [94]. Such recognition events mainly occur at the apoplast/outer membrane interface, where plant pattern recognition receptors (PRRs) are localized [95]. Upon perception, plants activate defense responses in qualitative and quantitative manners; in general,a higher concentration of MAMPs/DAMPs will result in the activation of more intense defense responses. The amplitude of such responses varies also depending on the type of molecule/epitope triggering the plant defense; to date, oligosaccharidescharides from plant cell walls with a proven nature of DAMPs (i.e., oligosaccharidescharins) include oligogalacturonides [10], cellodext [91; 96], and xyloglucan fragments [97]. Although the defense responses protect plants against microbial infections, hyperactivation of immunity negatively impacts plant development. Therefore, the uncontrolled _in planta_ expression of CWLEs may result in impaired growth, reduced productivity, and lethality [90; 98; 99]. In order to circumvent these undesired effects, different CWLEs expression strategies may be adopted, such as (i) compartmentalized expression/accumulation, (ii) inducible gene expression, (iii) inducible enzymatic activity, and (iv) use of plant hosts that are not sensitive to CWLE activity. Compartmentalized expression was attempted to constitutively accumulate CWLEs into different organelles such as chloroplasts [100; 101], lytic vacuoles [102; 103], and cytoplasm [104; 105], thus avoiding interaction with cell wall polysaccharides as well as the activation of PRRs that, in turn, may trigger immune responses (Figure 3a). In general, chloroplast-localized expression of CWLEs allows a high yield of recombinant protein to be obtained, even if, in some cases, chloroplast expression of cellulases has resulted in stunted growth and a pale-green phenotype [101]. The chloroplast expression system is not indicated for expressing fungal CWLEs that require glycosylation for proper activity and stability [106] and, more importantly, needs several rounds of selection for reaching homoplasmy (i.e., stable expression condition in which the plastomes of all chloroplasts are recombinant [107]). Alternatively, recombinant proteins may be targeted to vacuoles upon passage through the endoplasmic reticulum (ER) and the Golgi apparatus by fusing specific C-terminalopeptide sequences to the protein of interest [108; 109].