# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 4 Microbial Production
### Biotransformation
#### 4.1.1 Yarrowia lipolytica
An initial concentration of 60 g/L of castor oil followed by two additions of more than 60 g/L was the stepwise fed-batch strategy used. A maximum titer of 7 g/L was achieved after \(\sim\)80 h. This represented a 2-fold increase in comparison with the batch culture. The improvement of \(\gamma\)-decalactone production by the MTLY40-2P in comparison with the parental strain can be mostly justified by the elimination of the reconsumption of \(\gamma\)-decalactone due to the deletion of _YIPOX3_[65]. The oxidative state of the medium also influences biotransformation processes. For instance, it was shown that in a 2 L bioreactor using methyl ricinoleate as substrate the best strategy was an air flow rate of 0.9 vvm, and a pressure of 0.1 MPa (the lowest tested) in order to obtain a superior production of \(\gamma\)-decalactone (\(\sim\)300 mg/L from 10 g/L substrate). Moreover, the O\({}_{2}\) in the medium should be decreased when the titers of \(\gamma\)-decalactone were high [66]. The effect of oxygen and \(k_{\mathrm{L}}a\) on lactone production by _Y. lipolytica_ was a subject of other studies [67]. However, increasing the oxygen transfer rate through the agitation and aeration rate (to 650 rpm and 5.1 L/min) in a 3.7 L stirred bioreactor positively affected \(\gamma\)-decalactone production. The highest titers were obtained (5.4 g/L) using 60 g/L of cells and substrate (castor oil). In this case, similar productivities were observed for a batch and stepwise fed-batch (\(\sim\)215 mg/L/h), indicating that the first option was more suitable for the process and that at these concentrations of cells and substrate, castor oil is not toxic for _Y. lipolytica_[65]. Recently, the first step towards a more sustainable biotransformation process was given in _Y. lipolytica_. Hydroxy fatty acids are not an abundant and renewable substrate and therefore do not fulfill the needs of a circular bioeconomy. The case of ricinoleic acid present in castor oil is paradigmatic. It has been the main substrate used for biotransformation processes. However, castor oil has high toxicity associated with its extraction procedure. Castor oil is extracted from the castor beans of the native _R. communis_ plant [18]. Ricin is a very lethal toxin present in this plant, its effects being particularly relevant via inhalation. Moreover, due to the presence of this toxin but also to climate adaptability issues and labor-intensive harvesting processes, it is very difficult to pursue its plantation in developed countries [19]. Therefore, [PERSON] and colleagues engineered _Y. lipolytica_ to harness this yeast with the ability to convert non-hydroxy fatty acids into lactones [20]. As a proof of concept, oleic and linoleic acid were selected as substrates due to their high presence in low-value feedstocks, such as used cooking oil or animal fats. The yeast was engineered with different bacterial hydrates: an oleate 10-hydratase from _Stentrophomonas maltophilis_ to convert oleic acid into 10-hydroxysteratic acid, and a linoleic 13-hydratase from _Lactobacillus acidophilus_ to convert linoleic acid into 13-hydroxyoleic acid. The heterologous expression of theses enzymes allowed for the production of \(\gamma\)-decalactone from oleic acid and \(\delta\)-decalactone from linoleic acid. Further metabolic engineering with heterologous long-chain-specific oxidases and process engineering allowed for the production of 282 mg/L \(\gamma\)-dodecalactone after 110 h by one of the engineered strains in the fed-batch strategy [20]. Despite the lower titers when compared to traditional biotransformation processes of hydroxy fatty acids, this is a much-needed step towards more sustainable production of lactones.