# Microbial Biosynthesis of Lactones: Gaps and Opportunities towards Sustainable Production
## 4 Microbial Production
### Biotransformation
#### 4.1.1 Yarrowia lipolytica
Another strategy used to engineer this pathway involved the deletion of _YIPOX3/5_ while integrating multiple copies of _YIPOX2_ in the genome. In comparison with the wild-type, this strain produced more \(\gamma\)-decalactone from methyl ricinoleate (150 mg/L vs. 71 mg/L), had a lower production of unwanted lactones (3-hydroxy-\(\gamma\)-decalactone and decenolides) and almost did not reconsume \(\gamma\)-decalactone. However, its production was slower (80 h vs. 12 h) and the strain was not able to consume all the methyl ricinoleate present in the medium (3.5 g/L in 500 mL baffled shake flasks) [57]. Later on, a similar engineering strategy was applied to an industrial _Y. lipolytica_ strain (CGMCC accession number 2.1405) rather than the model strain _Y. lipolytica_ W29 (ATCC 20460) [58]. This diploid strain had the two _YIPOX3_ alleles deleted and multiple copies of _YIPOX2_, and was able to produce \(\sim\)3.3 g/L \(\gamma\)-decalactone from 50 g/L of methyl ricinoleate after 110 h. This represented a 3.6-fold improvement in comparison to the parental strain, an improvement superior to that observed in the W29 strain [57]. This study highlighted the importance of screening the intraspecies (strain-level) heterogeneity of microorganisms in microbial production no matter the process or product sought. Manipulation of the _YIPOX_ genes was the first and most effective strategy adopted to significantly improve biotransformation processes for the production of lactones in _Y. lipolytica_. The lipase of _Y. lipolytica_ also played a role, namely to improve the release of ricinoleic acid from castor oil. [PERSON] and colleagues (2012) [59] tested the effect of the addition of a lipase inducer (olive oil) in baffled-shake-flask fermentations using 30 g/L of castor oil for the production of \(\gamma\)-decalactone. Although the maximum production level was achieved earlier in the medium with the inducer (140 h vs. 185 h), the titers obtained were lower--1.2 g/L in the medium with inducer vs. 1.6 g/L in the medium without inducer. The productivities of the two conditions tested were similar [59]. Addition of commercial lipases to hydrolyze castor oil was also attempted with similar results; when commercial lipase was present the production of \(\gamma\)-decalactone was faster but presented lower titers [60]. Another important factor taken into account was the toxicity of lactones towards the microorganism [24]. \(\gamma\)-decalactone, \(\gamma\)-dodecalactone and \(\delta\)-decalactone were toxic for _Y. lipolytica_ in the range of 150-300 mg/L, negatively impacting the lag phase or even completely blocking growth. From those lactones, \(\gamma\)-dodecalactone produced the more pronounced negative effects [61]. The effect on the fluidization of the cellular membrane and interaction of the lactones with its components may, at least partially, explain the mechanism of toxicity [62; 63]. The optimization of process conditions for biotransformation employing _Y. lipolytica_ was also frequently sought, namely the operation mode of the reactor. An intermittent fed-batch strategy for the conversion of methyl ricinoleate was tested in _Y. lipolytica_. The strategy consisted in the addition of methyl ricinoleate at specific time points to avoid substrate-related toxicity. This approach was superior to a constant feeding strategy in terms of titers, productivity and yield. In comparison with a batch strategy, the productivity was lower but the titer and yield were superior. Overall, in this work the production of 6.8 g/L of \(\gamma\)-decalactone after \(\sim\)168 h was achieved [64]. Again, a stepwise fed-batch was the best operation mode for the production of \(\gamma\)-decalactone by an engineered _Y. lipolytica_ strain [65]. The MTLY40-2P strain had the _YIPOX2-5_ native genes deleted and the _YIPOX2_ gene overexpressed under the regulation of a promoter that allowed a decrease in catabolic repression. A cell density of 30 g/L was used for biotransformation of under 650 rpm and an aeration rate of 5.1 L/min.