## 2 Production of Lipases
### Production of Recombinant Lipases
#### 2.2.2 Eukaryotic Expression Systems
Those properties can potentially reduce production costs since these nutrient sources can be found in many industrial by-products. There are several reported strategies to increase the production and secretion of different lipases in _K. phaffii_. Examples and descriptions of some of the most applied strategies are presented in the paragraphs below. Promoters play an important role in the first step of setting up a cell factory for effective production of a desired protein. Different types of promoters, such as inducible and constitutively active ones, are available for heterologous gene expression in _K. phaffi_[94]. One of the most commonly used promoters is the inducible alcohol oxidase 1 gene promoter (pAOX1). The enzyme expressed by the pAOX1 is responsible for the conversion of methanol to formaldehyde, which is the first reaction in the metabolism of methanol. The promoter is tightly regulated by the presence of repressing carbon sources, such as glucose, glycerol and ethanol. To become fully activated, it requires the presence of methanol [95]. The pAOX1-based protein expression system can be easily applied to control bioreactor cultivations. The tight regulation by the repressing carbon source can enable cell growth to high densities prior to heterologous protein production. However, on an industrial scale this system is hazardous because methanol is a flammable chemical. For that reason, there have been attempts to identify or create promoters that achieve equal or even better protein yields [94; 96]. Another inducible promoter that has shown promising results is the formaldehyde dehydrogenase 1 gene promoter (pFLD). The formaldehyde dehydrogenase 1 enzyme is involved in both methanol metabolism and in the assimilation of some amines (ex. methylamine) as a nitrogen source. pFLD can be activated by methanol or methylamine and, similar to pAOX1, be repressed by some carbon sources. Cultivations using sorbitol as carbon source and methylamine as nitrogen source demonstrated that sorbitol did not repress the expression of enzymes involved in methanol metabolism, as formaldehyde dehydrogenase 1 [87]. It should be considered that inducible promoters require longer fermentation runs due to the induction step. Therefore, constitutive promoters may serve as a tool to create more cost-effective fermentation cultivations. One promoter that has been applied successfully to lipase production is the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Since it is a strong constitutive promoter, no induction step is required, resulting in simpler and shorter fermentation cultivations. The expression of _Fusarium solani_ NAN103 lipase (FSL) under pGAP or pAOX1 showed that pGAP-FSL produced the highest specific lipase activity and, in addition, there was a two-day reduction in the cultivation time [88]. Many promoters with different strengths and properties are available for expression in _K. phaffii_. Testing different types of promoters for constructing a specific protein cell factory is a good starting point for the further development and optimization of the heterologous expression system. With respect to lipase production, a promoter that gives good yields for one enzyme will not necessarily give the best results for another [92]. In most cases, increasing the copy number of a desired gene enhances the expression of the recombinant protein. In _K. phaffi_, the production of the lipase CALB was improved 2.3 fold by increasing the copy number to three. In this experiment, a defective marker approach was applied based on the leucine marker _leu2-d_, in which multiple copies were necessary to reach a state of prototrophy [65]. However, in some cases a high gene dosage can negatively affect the production because the secretory pathway may become overwhelmed by the effects of high levels of recombinant protein that need to be correctly folded [97]. When _R. oryzae_ ROL was expressed in up to eight copies, the best results were achieved with the strain harboring five copies, with a fold increase of eight.