# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 4 Microbial Production
### De Novo Biosynthesis
Several years later, solid-state fermentation of sugarcane bagasse employing this fungus led to the production of six different lactones: 6-pentyl-\(\alpha\)-pyrone, \(\delta\)-octalactone, \(\delta\)-dodecalactone \(\gamma\)-nonalactone, \(\gamma\)-undecalactone and \(\gamma\)-dodecalactone. Interestingly, \(\gamma\)-decalactone, the major lactone produced by almost all the organisms, was not produced by this fungus. Instead, 6-pentyl-\(\alpha\)-pyrone was the most produced lactone (3.62 mg per g of dry matter--equivalent to biomass) from a medium based on 4.5 g sugarcane bagasse after 5 days of culture [12]. The filamentous fungus _F. poae_ (Peck [PERSON] strain) is another of the few filamentous fungus reported to de novo biosynthesize lactones [34]. Eight saturated (\(\gamma\)-valero-, \(\gamma\)-capro-, \(\gamma\)-hepta-, \(\gamma\)-octa, \(\gamma\)-nona-, \(\gamma\)-deca-, \(\gamma\)-undeca and \(\gamma\)-dodecalactone), and one unsaturated (cis-6-\(\gamma\)-dodecenolactone) \(\gamma\)-lactones together with one \(\delta\)-lactone (\(\delta\)-decalactone) were detected in liquid cultures of this fungus using malt broth as a medium. Cis-6-\(\gamma\)-dodecenolactone was the major lactone produced (\(\sim\)2 mg/L) and the main contributor to the fruity aroma felt. This lactone was even possible to detect in solid cultures, while all others were only detected in liquid cultures after 3 days of growth. Even-chain lactones were more abundant than odd-chain lactones, and aeration of the culture was crucial for production. The fungus metabolism shifted from lactone to alcohol biosynthesis when insufficient aeration conditions were provided [34]. This fungus did not produce the same enantiomers for all the lactones detected [82]. In the case of the major lactone produced, cis-6-\(\gamma\)-dodecenolactone, the (R)-enantiomer was predominant. However, the (S)-enantiomer was described as more intense, despite having similar odor descriptors. This is a difference to what was observed, for instance, in _P. guillermondii_[71]. Further investigation on de novo biosynthesis of lactones by a different strain of _F. poae_ demonstrated that the addition of yeast extract and amino acids increased their production [83], but the major lactone produced continued to be cis-6-\(\gamma\)-dodecenolactone. It was hypothesized that its synthesis started from an oxygenated derivative of linoleic acid, the major unsaturated fatty acid accumulated by the fungus. Thus, hydroperoxides, epoxides, and hydroxy derivatives from unsaturated fatty acids were considered to be the potential precursors of lactones [83]. This hypothesis was later reinforced not only by the numerous biotransformation processes developed but also by studying different organisms that are able to de novo biosynthesize lactones, such as fruits [21; 22] and other fungi [23]. _Penicillium roqueforti_ was another filamentous fungus used to study the complexity involved in the biosynthesis of lactones. Spores of this fungus were able to convert soybean fatty acids (mainly oleic and linoleic acid) predominantly into \(\gamma\)-dodecalactone and cis-6-\(\gamma\)-dodecenolactone [84]. The conversion of oleic acid into \(\gamma\)-dodecalactone was consistent with the action of a 10-hydratase, which introduced a hydroxy group at C10. Regarding cis-6-\(\gamma\)-dodecenolactone, the post-modification of linoleic acid appeared to be the action of a 10-lipoxygenase. These first reactions were followed by three sequential cycles of \(\beta\)-oxidation. Despite these proposals, these enzymatic steps were not genetically or experimentally validated [84]. Since the 1970s there have been reports on the ability of fungi to de novo biosynthetic different lactones [4]. These works identified the ability of a species/strain to de novo biosynthesize lactones in different culture conditions and used chromatography-mass spectrometry technology to disclose the chemical configuration of the lactones produced and their precursors.