# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 4 Microbial Production
### De Novo Biosynthesis
Some important insights were advanced, such as the hypothesis of the lactones being produced by derivatives of unsaturated fatty acids [83], which was the basis for the development of biotransformation processes. However, those studies added scarce information about the metabolic or physiological mechanisms of the de novo biosynthesis of lactones [41]. Additionally, there was no attempt to improve the ability of the microorganisms to de novo biosynthesize lactones either by a lack of molecular/bioprocess tools or because biotransformation offered higher titers and was a more promising alternative for the development of a competitive process. In this matter, the identification of _A. gossyspii_ as a fungus able to de novo biosynthesize lactones from sugars was important [23; 85]. This fungus can synthesize several \(\gamma\)-lactones. Nine different wild strains of the fungus were able to de novo biosynthesize up to seven \(\gamma\)-lactones, namely \(\gamma\)-valero-, \(\gamma\)-capro-, \(\gamma\)-octa-, \(\gamma\)-nona-, \(\gamma\)-deca-, \(\gamma\)-undeca- and \(\gamma\)-dodecalactone [23]. From these, \(\gamma\)-octa-, \(\gamma\)-nona-, \(\gamma\)-deca- and \(\gamma\)-dodecalactone were detected at titers above their aroma perception threshold. \(\gamma\)-decalactone was the major lactone produced in the range of mg/L from a glucose-based medium after 2 days [23]. More importantly, the work of [PERSON] and colleagues was the first to report the improvement of the de novo biosynthesis of lactones by metabolic engineering. Having oleic acid as the central fatty acid, the authors addressed the de novo biosynthesis of lactones by identifying two main modules for improvement: (i) the synthesis of fatty acids from carbohydrates, comprising the fatty acid biosynthetic pathway and the desaturation and elongation system, and (ii) the conversion of the fatty acids into lactones, comprising the \(\beta\)-oxidation pathway and the lactonization step (Figure 2). Thus, strains were engineered with the following genetic modifications: (i) overexpression of _AgDES589_, to promote the conversion of oleic acid into other derivatives, namely linoleic acid; (ii) the deletion of _AgELO624_, which encodes an elongase that catalyzes the formation of very long fatty acids (C20:0 and C22:0) that are not interesting for lactone biosynthesis; and (iii) the substitution of the _AgPOXI_ gene, encoding the native acyl-CoA oxidase, for the heterologous _YIPOX2_, which encodes a specific long-chain acyl-CoA oxidase from _Y. lipolytica_, in order to fine-tune the biosynthesis of lactones towards \(\gamma\)-decalactone (C10). These engineered strains increased total lactone biosynthesis by more than 6-fold, specially \(\gamma\)-decalactone, in comparison with the parental strain in glucose-based media during cultures of 72 h [23]. This was the first work reporting the engineering of a microbial host for improving the de novo biosynthesis of lactones. This modular approach indicated that the biosynthesis of lactones can be engineered, not only by improving the conversion of the fatty acids into lactones, but also by engineering the fatty acid biosynthesis to increase the production of the desired fatty acids. In sum, by accomplishing the enhancement of microbial de novo biosynthesis of \(\gamma\)-lactones through engineering of the host, this work paved the way for the bioproduction of lactones from sustainable substrates. The work of [PERSON] and colleagues was also important in highlighting the role of a desaturase encoded by the gene _AgDES589_, which was previously characterized to be responsible for the conversion of oleic acid into linoleic acid [86]. Overexpression of _AgDES589_ in _A. gossyspii_ led to a significant increase in the biosynthesis of lactones, mainly \(\gamma\)-decalactone, confirming the importance of this enzyme [23]. No orthologs of this gene are present in _S. cerevisiae_ or in other post-whole-genome duplication (WGD) species of the Saccharomycesacetae, which may justify why _A.