## 2 Production of Lipases
### Production of Recombinant Lipases
#### 2.2.2 Eukaryotic Expression Systems
Since strains harboring more copies produced lower levels of ROL, these strains may have potentially produced unfolded proteins that lead to ER stress and the activation of the unfolded protein response (UPR) [86]. Therefore, it is important to adjust the copy number by taking into account the protein folding capacity of the host. Even though _K. phaffii_ has the potential to fold lipases properly, the native system's secretory pathway may reach its full capacity. This might result in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) that will trigger the activation of UPR. In this case, the UPR functions by reducing the translation of proteins, degrading misfolded proteins and increasing the production of molecular chaperones involved in protein folding. There are many different types of chaperones with various refolding and/or stabilization properties [98]. As an example, protein disulfide isomerase (PDI) catalyzes the formation and breakage of disulfide bonds between cysteine residues, permitting proteins to achieve their correct folding. Other chaperones, such as Kar2p, also known as binding immunoglobulin protein (BIP) in mammals, play key roles in protein translocation through the ER. BIP/Kar2p stabilizes immature proteins by binding to their hydrophobic amino acids, allowing the protein to achieve its correct folding. Moreover, BIP/Kar2p prevents unfolded protein aggregation by assisting in ER-associated degradation (ERAD). The overexpression and co-overexpression of chaperones in _K. phaffii_ have been demonstrated to improve protein secretion and production yield [99]. The co-expression of PDI increased the production of the MAS1 lipase from marine _Streptomyces_ 1.7 fold. MAS1 was expected to have two pairs of disulfide bonds and the higher levels of PDI were predicted to assist their formation [83]. Interestingly, increasing the copy number of PDI does not always automatically increase the protein production and secretion. _Rhizopus chinensis_ lipase r27 RCL was co-expressed with different copy numbers of PDI in _K. phaffii_. PDI was expected to ease bottlenecks in the ER, since the crystal structure of the lipase r27 RCL revealed that it contains three disulfide bonds. One copy number of PDI resulted in a one fold increase in enzyme activity, whereas two, three and four copies resulted in lower activity. The higher copy number of PDI may reduce the availability of transcription and translation machinery for the expression of the lipase gene [84]. The overexpression of different combinations of chaperones has also resulted in an increase in lipase production. The production of the lipase CALB from _C. antarctica_ was tested with the individual overexpression of different chaperones (Ydj1p, Ssa1p, Sec63p, and Kar2) and with a combination of them. The chaperones Ssa1p and Ydj1p stabilize the nascent peptide chain and help its translocation into the ER. Sec63p binds to the translocation peptide and prevents it from moving backwards through the ER channel. The best results were achieved with the combination of Ydj1p-Ssa1p, increasing the production by 2.5 fold. This suggests that these chaperones interact synergistically by enhancing protein folding, secretion and preventing protein aggregation. In contrast, a decrease in CALB was observed with the expression of Kar2p. This could indicate that the excessive presence of Kar2p shifted the ER balance towards the ERAD pathway, resulting in decreased CALB secretion [85]. Alternative Eukaryotic Hosts  
Different hosts can provide a range of diverse properties, which may be better suited to a specific cell factory design. For that reason, a lot of effort has been made in recent years to find other eukaryotic hosts for lipase production. The yeast _Y. lipolytica_ is considered a suitable production host because of its expanding genetic tools for heterologous expression, and its ability to metabolize raw substrate and sectre protein in large amounts [100]. The comparison of CALB production by _Y. lipolytica_ and _K.