4 Discussion
We have evaluated the performance of seven FADs and four FARs, which are involved in insect sex pheromone biosynthesis, to establish the production of Z 11-14:OH in Y. lipolytica . Among FADs, Lbo_PPTQ showed the highest production of (Z )–11–tetradecenoate and was selected for further strain engineering. Several desaturases, namely, Onu11, EpoE11, and CpaE11, were not active. The desaturase Onu11 was previously assayed in other recombinant hosts, such as S. cerevisiae and Nicotiana benthamiana with divergent results. In S. cerevisiae , it generated E /Z 11-14:acid in ratio 4:5 and also producedZ 11-16:acid [26], while no activity of this enzyme was observed in N. benthamiana [14]. The other two desaturases that were non-functional in our study, EpoE11 and CpaE11, were reported to act on 14:acid and produce E 11-14:acid inS. cerevisiae , but the amounts were very low [29,31]. One possible explanation is that the non-functional desaturases were poorly expressed or misfolded. A recent study by Buček et al. showed that different moth desaturases could have different expression levels [38]. Codon-optimization could also have been suboptimal [39].
After examining FADs, we screened several FARs for increased specificity towards fatty acids with C14 chain length. Previously, HarFAR was shown to prefer (Z )–9–tetradecenoate over (Z )–11–hexadecenoate and (Z )–9–hexadecenoate [34]. We hypothesized that this reductase should also be able to act on (E /Z )-11- tetradecenoate, which is structurally similar to (Z )-9-tetradecenoate. Indeed, a feeding experiment and co-expression with Lbo_PPTQ confirmed that HarFAR could produceE /Z 11-14:OH from corresponding acids. (E /Z )-11- tetradecenoate was also converted into alcohol by Y. lipolytica expressing SlitpgFARII. This FAR previously produced the highest amounts of Z 11-14:OH among the four tested reductases from Spodoptera spp. [35]. OnuFAR_E and OnuFAR_Z were shown to be selective for E and Z isomers of 11-tetradecenoate, respectively [36], however, in our study, OnuFAR_E showed little and OnuFAR_Z no activity. The same results were obtained when these enzymes were screened in the plant N. benthamiana [14]. One possible explanation for the lack of functionality of these enzymes in yeast and plant expression hosts could be incorrect folding of the reductases in the ER membrane, which has a different lipid composition in yeasts and plants than in insects [40-43].
In order to improve the production of Z 11-14:OH, we employed metabolic engineering strategies, such as integration of multiple copies of genes and enhancement of precursor supply by overexpression ofFAS1 subunit of Y. lipolytica . In small-scale cultivations of the engineered yeast strain, we achieved 93.9 ±11.7 mg/L ofZ 11-14:OH. On the way of building the production strain (ST9253), we observed that introduction of desaturase Lbo_PPTQ not only contributed to the biosynthesis of Z 11-14:OH, but it also improved the total fatty alcohol titer. It was previously shown that overexpression of the native OLE1 desaturase increased fatty acid biosynthesis in general, likely because the fatty acid synthase complex FAS is less inhibited by unsaturated fatty acyl-CoAs than by saturated fatty acyl-CoAs [44-46].
For collection of pheromone for EAG and behavioral tests, the engineered strain was fermented in controlled 1 L bioreactors in fed-batch mode. We used glycerol as the carbon source with a high carbon-to-nitrogen ratio to favor fatty alcohol production [47-52]. We obtained 188.1±13.4 mg/L of Z 11-14:OH. The strain also produced large amounts (over 1.3 g/L) of the saturated by-product 14:OH, indicating a significant limitation of the desaturation step. This may be improved by further strain engineering and fermentation optimization in the future. To our knowledge, this is the first study showing the production ofZ 11-14:OH in a microbial host. Previously this pheromone precursor was recombinantly synthesized in plant N. benthamianayielding 14 µg from 1 g leaf tissue [14]. While Y. lipolyticahas been engineered for the production of fatty alcohols in multiple other studies, the common products are naturally unsaturated or saturated fatty alcohols [48,49,53-56].
To convert the fermented alcohol into acetate, which is the active sex pheromone component of O. nubilalis , a chemical acetylation step was performed, which resulted in full conversion and yielded 320±13.4 mg of Z 11-14:OAc. Interestingly, based on the current knowledge about insect pheromone biosynthesis, this reaction should be catalyzed by acyltransferases in insects [6,57]. However, until now, no enzymes from moths catalyzing this reaction have been found even though some gene candidates have been proposed and tested [58].
The yeast-derived pheromone blend caused a response of O. nubilalis males in electroantennogram experiments similar to what could be expected based on the responses to synthetic pheromone compounds. Furthermore, the blend was attractive to insects in behavioral bioassays in a wind tunnel to the same level as the chemically synthesized pheromone blend. However, full precopulatory behavior was observed less often. In order to induce a complete the ECB precopulatory behavior, a higher purity may be required, as certain by-products present in the blend may hinder the complete expression of the precopulatory behavior. Notably, reduced precopulatory behavior is not necessarily a hindrance and may even be a benefit for mating disruption. Therefore, activity studies in the field are warranted.
In summary, we have successfully employed yeast Y. lipolytica for production of O. nubilalis sex pheromone precursorZ 11-14:OH and showed that the resulting yeast-derived pheromone was biologically active in modulating the behavior of O. nubilalis males.