# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 5 Towards Sustainable Production of Lactones: Final Remarks and Perspectives  
Lactone biosynthesis in natural producers, such as fungi and fruits, is a complex and multi-pathway process (Figure 2). From the current knowledge, it starts with the biosynthesis of fatty acids, specifically the biosynthesis of unsaturated fatty acids, which already involves enzymes from the desaturase system of the host [23]. Engineering the fatty acids biosynthetic pathway with the purpose to increase the biosynthesis of lactones had not been done by the date of this review. However, choosing a microbial host that possesses a good ability to synthesize fatty acids (e.g., oleaginous yeasts) and a good molecular toolbox to fine-tune the type of fatty acid that it accumulates could be the first step to create improved chassis strains. In addition, microorganisms that are able to do this in different carbon sources (glucose, glycerol, xylose, etc. ), such as those present in industrial wastes or renewable raw materials (lignocellulosic biomass or crude glycerol), will be preferred. Afterwards, three additional stages are needed to biosynthesize a lactone. The first is the introduction of a hydroxy group to the fatty acid, which can be achieved in one step or through more than one enzymatic reaction. The position of this hydroxylation in an even or odd position will be determinant to the generation of a \(\gamma\)-lactone or \(\delta\)-lactone, respectively [6; 11]. Several enzymes were proposed for this end. Until the date, the most biochemically promising seems to be hydratases [20; 78] and desaturases/hydroxylases [23], with reported results for the production of lactones in _Y. lipolytica, W. lipofer_ and _A. gossypii_. The source of these enzymes is diverse, from bacteria to fruits. Regardless, these sources of genetic material need to be tested in industrial microorganisms in order to study and improve microbial production of lactones. The fact that some desaturases identified in fruits and microorganisms share high homology with other fatty acid modification enzymes, such as hydroxylases and epoxidases, is also noteworthy [21; 23]. Indeed, there are cases of enzymes that can catalyze both hydroxylation and desaturation, and in which the change of a few specific amino acids can alter their function [21]. Therefore, it is important to identify and characterize these bifunctional enzymes. The enzymes responsible for the chemical transformation of fatty acids before entering \(\beta\)-oxidation are the crucial and most complex distinguishing factor between microorganisms that perform de novo biosynthesis of lactones or only biotransformation. The next step of chain shortening in the \(\beta\)-oxidation pathway is the most well-studied stage. As described above, this pathway was comprehensively studied in _Y. lipolytica_[24]. An acyl-CoA oxidase from this yeast was used with success to improve lactone biosynthesis in _A. gossypii_[23]. Nonetheless, other acyl-CoA oxidases should still be tested, providing better results, as is the case of plant-derived oxidases in _Y. lipolytica_[20]. Lastly, lactonization is the step that is more uncontrollable and poorly known from a biosynthetic point of view. During fermentation, it occurs with high frequency in acidic conditions, and therefore it is thought that lactonization is spontaneous rather than enzymatic, since no enzyme was identified so far. Moreover, it is unclear whether lactonization occurs completely intracellularly or if the hydroxy acid precursors are excreted and undergo spontaneous lactonization in the extracellular medium [11]. However, the identification of candidate genes in peaches opens good perspectives for the future [21]. In addition, it would be interesting to test enzymes similar to those in olefinic hydrocarbon biosynthesis, which are able to perform the conversion of \(\beta\)-hydroxy acids into \(\beta\)-lactones [90].