# 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 Fungi and Bacteria  
To date, different bacteria and yeast species have been used as expression hosts for CWLE production. These microorganisms differ significantly in their cell wall structure and subcellular compartments, thus affecting both protein secretion and expression efficiency. Over the past years, expression of both single CWLE and enzyme mixtures has been performed in different bacteria species (i.e., _Bacillus_[40] and _Clostridium_ species [41] as well as _E. coli_[42; 43]). Constitutive promoters were exploited to maximize the expression of many cellulases [44; 45; 46; 47], avoiding the additional cost from the use of chemical compounds as transcriptional inducers. However, side effects occurred in some cases, as the saturation of transmembrane transport mechanisms had inhibitory effects on cell growth and viability [46; 47; 48]. To reduce such toxic effects, weakening the promoter strength through mutagenesis was of help [46; 48]. Alternatively, cellulases were expressed under the control of a growth phase specific inducible promoter [47; 49]. Moreover, to overcome inefficient expression caused by the different codon usages of the various bacterial species, synthetic genes were codon-optimized according to the codon usage of the expression host [45; 50]. Although these molecular tools have led to significant progress, efficient heterologous expression of secreted CWLEs from bacterial hosts is still challenging. The low levels of soluble proteins and the poor secretion ability represent the main limitations of Gram-negative bacterial hosts. Although a number of secretion pathways have been elucidated in the model organism _E. coli_[51], the translocation step across a double membrane system negatively impacts the yield of secreted CWLEs [52]. Different strategies have succeeded in increasing the secretion efficiency of heterologous proteins from _E. coli._ For example, the solubility of some cellulases was enhanced (i) by fusing their GH domain to the CBM from another (highly soluble) cellulase [53], (ii) by replacing the native signal peptide with those from efficiently secreted proteins [44; 47; 54], or (iii) by expressing synthetic sequences optimized with the codon-usage of the expression host [55]. Other approaches allowed the purification of active cellulase and hemicellulose from inclusion bodies by using denaturing agents such as urea and \(\beta\)-mercaptoethanol coupled to a subsequent refolding step [56] or by reducing the temperature of expression in order to ensure correct protein folding [57]. Alternatively, different species, especially those belonging to Gram-positive bacteria (e.g., _B. subtilis_ or _L. lactis_), were selected as expression hosts since they secrete the recombinant proteins more efficiently than Gram-negative bacteria [58; 59]. In eukaryotic organisms, yeasts are eligible hosts for overcoming the solubility and secretion limits that affect heterologous expression in the bacterial system. Several yeasts such as _Saccharomycescerevisiaee, Pichia pastoris, Kluyveromyces lactis, and Yarrowia lipolytica are characterized by high expression yields and the ability to perform eukaryotic post-translational modifications (i.e., disulfide bond formation and N-glycosylation), which are often present in CWLEs, therefore enhancing their stability and activity. CWLEs not efficiently produced in bacterial hosts can be secreted in active forms from yeast, as in the case of the endo-pgA from _Aspergillus niger_ that failed to acquire proper folding upon expression in _E. coli_; conversely, the active form of endo-pgA was successfully secreted in high amounts by _Saccharomyces cerevisiae_[60]. Many CWLE-encoding genes have been successfully expressed as secreted proteins in _S. cerevisiae_, representing the host of choice for expressing a variety of cellulases [61; 62], hemicellulases [63; 64], and pectinases [60]. Another well-described and widely applied yeast for the heterologous expression of CWLEs is the methylotrophic yeast _P. pastoris_[65].