# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 3 Pathways and Enzymes Involved in the Biosynthesis of Lactones
### Fruits  
Fruits like peaches and strawberries are intense producers of lactones, mainly during ripening. These compounds largely contribute to the flavor and aroma of these fruits. Of the more than 100-360 volatile aroma compounds previously identified, lactones have the most pronounced effect on the fruit's aroma perception. Thus, since their genome was sequenced, these organisms have aided the elucidation of the intricate biosynthesis of lactones [21; 22]. It is clear that the process starts from the fatty acid metabolism. This assumption is reinforced by the fact that in peaches, fatty acids and lactones are inversely regulated, that is, lactones increase during ripening while fatty acids decrease, indicating that the latter are the source of the former [21]. These precursors undergo some post-modifications in order to be converted into a hydroxy fatty acid before entering the \(\beta\)-oxidation, where the immediate precursors of lactones are formed: 4- or 5- hydroxy acids. In fruits it is also not understood whether the last step of lactonization or cyclization occurs completely by spontaneous reaction or if it is catalyzed by an enzyme. In this regard, a gene from peaches that encodes a novel type of alcohol acyl transferase was proposed as a good candidate for this last step [21]. The introduction of the hydroxy group in fruits seems to occur similarly to what happens in microorganisms. In this matter, important enzymes that can potentially catalyze the formation of hydroxy fatty acids were identified. Back in the 1990s, even when no whole genomic data was available, it was reported that in peaches (_Prunus persica_ var. _nucipersica_) and strawberries (_Fragaria ananassa_) epoxidations of polyunsaturated fatty acids (e.g., linoleic or linolenic acid) were the source of oxygenated derivatives used for the biosynthesis of lactones [48]. In peaches, candidate genes for each of the following proposed reactions, which are stated to be the route from unsaturated fatty acids to hydroxy fatty acids, were identified: (i) epoxidation by an epoxidase, a peroxygenase or a cytochrome P450 (CYP) followed by the action of an epoxide hydrolase; (ii) direct hydroxylation catalyzed by a CYP or a hydroxylase; and (iii) desaturation by a desaturase followed by a hydration catalyzed by a hydratase or a CYP [21]. In strawberries, one of the most promising candidate gene is _FaFAD1_ (_Fragaria \(\times\) ananassa_), which encodes an enzyme similar to fatty acid desaturases that catalyze the conversion of oleic acid into linoleic acid. The expression of this gene strongly correlates to the biosynthesis of \(\gamma\)-decalactone in ripe strawberries. Surprisingly, FaFAD1p conserves some crucial amino acid residues of bifunctional desaturases/hydroxylases and is also closely related to a RcFAH12p hydroxylase present in _Ricinus communis_ castor beans--the plant that produces castor oil, which is the main source of the ricinoleic acid used for biotransformation processes [18]. Hydroxylases and desaturases share similar reaction mechanisms and therefore can be identified as homologs. This was the case of the oleate 12-hydroxylase from _R. communis_ L., which is a homolog of a fatty acyl desaturase [49]. Taken together, it was proposed that \(\gamma\)-decalactone synthesis in strawberries goes from the conversion of oleic acid into linoleic acid via the desaturase activity of FaFAD1p. Linoleic acid will then be converted into a hydroxy fatty acid (ricinoleic acid or derivative) via the action of FaFAH1p, a CYP-like enzyme also associated with the production of this lactone. Alternatively, the hydroxylase activity of FaFAD1p would directly catalyze the hydroxylation of oleic acid to ricinoleic acid, generating the precursor for \(\beta\)-oxidation [22]. However, none of these proposed endogenous fatty acid modification enzymes have been experimentally demonstrated in fruits nor in any other model.