# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 4 Microbial Production
### Biotransformation
#### 4.1.1 Yarrowia lipolytica  
The most well-established biotransformation process for lactone production is clearly the production of \(\gamma\)-decalactone from castor oil, ricinoleic acid or derivatives by _Y. lipolytica_. Castor oil is extracted from the _R. communis_ plant and is very rich in ricinoleic acid [18]. These substrates are widely used but there are indications that switching castor oil for alkyl ricinoleates could help reduce the formation of foam and would be favorable for the downstream processing [6]. The biotransformation processes count with the good ability of this yeast to tolerate high concentrations of fatty acids and to perform its sequential shortening through \(\beta\)-oxidation (four cycles) before the formation of the immediate precursor of \(\gamma\)-decalactone, 4-hydroxydecanoic acid. In this process, only part of the lactonization occurs during the fermentation, being necessary the acidification and heating of the culture to achieve high yields. Normally, this process result in titers between 5-10 g/L \(\gamma\)-decalactone [6; 11]. [PERSON] and colleagues started the genetic engineering work with _Y. lipolytica_ to improve the biotransformation of hydroxy acids into lactones [53]. The peroxisomal \(\beta\)-oxidation is the pathway responsible for these production processes. It is composed of four reactions catalyzed by three different enzymes that, in a cyclic manner, remove two carbons from the chain of a fatty acid. The enzymes are an acyl-CoA oxidase, a multifunctional hydratase/dehydrogenase and a thiolase. In this work, the authors found that the deletion of one acyl-CoA oxidase encoded by _YIPOX1_ increased the activity of the pathway and reduced \(\gamma\)-decalactone production. This lower production level was attributed to the superior consumption of this lactone or of its immediate precursor (4-hydroxydecanoic acid) by the engineered strain. This led to the assumption that the production levels can be considered an equilibrium between the production and consumption of lactones, with \(\beta\)-oxidation playing a relevant role in the catabolism of aromas. On the other hand, overexpression of the thiolase (_YIPOTI_) did not result in any detectable effect [53]. In regard to \(\beta\)-oxidation, _Y. lipolytica_ seems to have a clear advantage in catabolizing different fatty-acid-derived compounds. In contrast to other yeasts that only encode 1 acyl-CoA oxidase, _Y. lipolytica_ encodes five (_YIPOX1-5_) [54]. These enzymes vary from each other regarding the chain-length-dependent activity. Concerning the two major oxidases, _YIPOX2_ encodes for a long-chain oxidase with activity towards C18-C11, while _YIPOX3_ encodes for a short-chain oxidase (<C11). A strain deleted in this short-chain oxidase, YIPOX3p, produced more than 4-fold \(\gamma\)-decalactone from methyl ricinoleate than the parental strain (220 vs. 50 mg/L) [55]. This significant increase could be due to the slower recomsumption of lactones or their precursors (e.g., 4-hydroxydecanoic) by the engineered strain, since \(\beta\)-oxidation was affected at the C10 level--its growth was clearly diminished in C10 substrates [54; 56]. In order to completely block this reconsumption, no oxidase activity should be present below C10. This would only be possible with the deletion of all _YIPOX_ genes except _YIPOX2_. However, the resulting strain would have serious growth defects on fatty acids [56]. In turn, YIPOX4p, identified as an oxidase of minor relevance when methyl oleate was used [54], was found to be essential for the biotransformation of methyl ricinoleate into \(\gamma\)-decalactone [55]. YIPOX1p does not contribute to the oxidase activity in this yeast. However, the results obtained by [PERSON] and [PERSON] (1998) [53] are justified by the fact that the deletion of one _POX_ gene resulted in increased activity of the other oxidases [54]. Finally, _YIPOX5_-encoded oxidase has broad substrate specificity [54].