# Green Production and Biotechnological Applications of Cell Wall Lytic Enzymes
## 6 Conclusions  
An ideal bio-factory of CWLEs should be characterized by a high yield of recombinant protein, sustainable manufacturing, and a reduced production time and cost. In order to select the most appropriate production platform, the type of CWLE to express and its biotechnological purpose represent additional factors to be considered. Although bacteria and yeasts can produce high yields of recombinant proteins in very short time, expensive fermentation procedures are required to obtain high-level expressions, negatively impacting the sustainability of the entire process. Furthermore, CWLEs from lignocellulosic fungi can be characterized by the presence of disulfide bonds and glycosylations; therefore, the expression in bacteria and yeasts could result in misfolded or hypergly enzymes, respectively. Importantly, the use of CWLEs from microbial biofatories in food processing, nutraceutical manufacturing, and the medical field implies expensive purification procedures and strict quality controls, since bacterial contaminants, as well as yeast glycosylation, may cause allergic reactions in humans. On the other hand, plant biofatories are both a cheaper and more eco-friendly alternative, and, noteworthy, they can guarantee proper post-translational modifications to the eukaryotic proteins. Moreover, they are GRAS organisms, allowing the obtainment of high-value products at reduced production costs. Although the plant production of CWLEs may affect the fitness of the expression host, mainly because of their intrinsic phytopathogenic nature, such deleterious effects can be avoided by the different expression strategies here reported. However, plants need arable land for their growth and longer times to produce significant amounts of recombinant proteins. Under this perspective, microalgae may represent the best compromise since their cultivation does not subtract arable lands from the agri-food sector, and they are characterized by faster growth rates compared to plants. It is worth noting that genetic manipulation of microalgae has been less investigated; therefore, further optimization is still required for improving the expression stability and protein yield of microalgal-based biofactories. In conclusion, the most appropriate CWLE expression host should be determined on a case-by-case basis, taking into account both the characteristics of the specific CWLE to express and its application field.  
Figure 1, Figure 3a,b, and Figure 4 were generated using Biorender ([[https://biorender.com/](https://biorender.com/)]([https://biorender.com/](https://biorender.com/))). In Figure 3c, the 3D structure of XynA from _Bacillus halolurans_ (PDB number 2 UWF) and from _Thermotoga maritima_ (PDB number 1 VBR) are reported with that from _Rizhoctonia solani_ (UniProtKB A0A0K6 FTH) as obtained by structure modeling using SWISS 2D model ([[https://swissmodel.expasy.org/](https://swissmodel.expasy.org/)]([https://swissmodel.expasy.org/](https://swissmodel.expasy.org/))). Electrostatic surface potentials were generated with CCP4 MG software (negative and positive residues are in red and blue, respectively).  
[PERSON] and [PERSON] identified patterns and trends in the literature and designed the structure of the review. [PERSON] and [PERSON] prepared the tables and figures. All of the authors contributed to searching for relevant literature, carried out a critical analysis of the literature, discussed together, and wrote the manuscript. All authors read and approved the final manuscript.  
This research was partially funded by the Italian Ministry of Education, University, and Research (MIUR) in the frame of the initiative \"Departments of excellence\".  
Because of space constraints, a number of noteworthy publications have not been cited or discussed properly. We apologize to the authors for this lack of completeness.  
The authors declare no conflicts of interest.