## 2 Production of Lipases
### Production of Recombinant Lipases  
When the natural lipase producer can be efficiently propagated in a bioreactor, homologous expression is an option. In this case, the process is often more straight forward as the production of the lipase may already be naturally optimized. Higher lipase yields are often desirable, and this can be achieved by using stronger promoters to drive the lipase gene. Moreover, the host can be equipped with additional lipase gene copies via plasmids containing the lipase gene or by inserting lipase genes into the genome. To this end, there are a few studies on the production of recombinant lipases via homologous expression in the bacteria _Serratia marcescens_[47] and _Burkholderia cepacia_[48; 49], and in the filamentous fungus _A. niger_[27]. However, native lipases may not be suitable for industrial applications, as they might not meet the technical requirements of the scale-up process, including fast growth, high levels of protein production, or optimal physiological properties required in bioreactors. Hence, heterologous expression is an alternative by using known efficient hosts. Technically heterologous expression systems comprise basically three steps: (i) cloning of the gene of interest in a vector having a selection marker, (ii) transformation of the host strain with the constructed plasmid, and (iii) expression of the gene of interest under the control of an inducible or constitutive promoter and a known terminator. The biological systems used for gene expression include prokaryotic and eukaryotic hosts, such as _E. coli_ and _K. phiffi_, respectively [41; 43; 44]. Heterologous expression has also been the choice of many studies of recombinant lipases produced by uncultured microorganisms from extreme environmental conditions. Their lipases encoding genes can be isolated and expressed in heterologous expression systems by the construction of functional metagenomics libraries [50; 51; 52]. Functional metagenomics has the advantage of not requiring either individual genome sequencing or the cultivation of unknown producer microorganisms. For more metagenomics studies of lipases with biotechnological potential, [PERSON] and collaborators [52] provide an interesting review. Regarding regulatory mechanisms of lipase biosynthesis, it has been observed that they broadly vary in different microorganisms. In the case of _Calvatia_[53], _Rhizopus_[54], _Aspergillus_[55], and _Rhodotorula_[56], apparently, lipase production is constitutive and independent of the addition of lipids, despite the fact that their presence enhanced the levels of lipase produced. Conversely, the literature suggests that long-chain fatty acids - for instance oleic acid - participate in lipase expression in _Geotrichum candidum_, by controlling induction in the transcription level [57]. Concerning carbon sources, in _Fusarium_ sp., carbohydrates have been described as repressors of lipase production [58]. Interestingly, in terms of comparison, the expression levels of lipase genes in some organisms, including _K. phiffi_ and _Aspergillus_, can be hundreds of times higher than that in the native host, resulting in levels even greater than grams per liter [59; 60; 61]. In addition to the construction of strains for overproduction of recombinant lipases, the fermentation conditions should be optimized in order to reach cost effective yields of productivity. The most relevant parameters are pH, temperature, nutrients (carbon and nitrogen sources and salts), oxygen and agitation. In recent decades, the use of multivariate analytic tools--i.e., response surface methodology (RSM), has allowed for the optimization of lipase production by investigating different parameters simultaneously, which allows higher yield levels compared to univariable studies [35]. A lipase from _Bacillus_ sp. was heterologously produced in _E. coli_ and the authors screened different media and observed that, when _E. coli_ was cultivated in Nutrient Broth, maximum extracellular lipase was achieved.