3.1 Screening of fatty acyl-CoA desaturases
In O. nubilalis , a ∆11 desaturase acts on myristoyl-CoA (14:CoA), which in turn is generated by β-oxidation of the abundant fatty acid synthesis product hexadecanoyl-CoA (16:CoA), and generates a mixture ofE /Z 11-14:CoA [6,26]. In Y. lipolytica , myristic acid is a minor component of the total fatty acid pool, comprising only about 0.25% of the total fatty acids [27]. To increase the supply of 14:CoA in Y. lipolytica , we substituted isoleucine 1220 of the fatty acid synthase ketoacyl synthase domain to phenylalanine (Fas2pI1220F). This mutation was proposed to hinder the binding of longer acyl-CoAs in the active site tunnel of the FAS complex and was reported to result in the production of shorter chain acyl-CoAs [27]. The mutation has already been proven to be effective in the production of Z 9-14:OH, where it increased the titer 15-fold [16]. In this study, we introduced the FAS mutation intoY. lipolytica strain ST6629, which was previously developed for decreased degradation of fatty alcohols and decreased storage lipid synthesis [16]. The resulting ST7982 showed 8.4-fold increase in myristic acid production compared to the parental strain (Supplementary Figure S1).
Next, seven ∆11 desaturases with previously documentedE /Z 11-14:CoA activity were tested in strain ST7982 (Figure 1, Figure S2). These were the desaturase Lbo_PPTQ from the grapevine moth Lobesia botrana [28], Onu11 from the European corn borerO. nubilalis [26], EpoE11 from the light brown apple mothEpiphyas postvittana [29], CroZ11 from the oblique banded leafroller moth Choristoneura rosaceana [30], CpaE11 from the spotted fireworm moth Choristoneura parallela [31], Hzead11 from the corn earworm H. zea [32], and Msextad11 from the tobacco hornworm Manduca sexta [33]. The resulting strains were cultivated, their lipids methanolized into FAMEs, and analyzed on GC-MS. Lbo_PPTQ expression resulted in the highest content ofZ 11-14:acid. The Z /E isomer ratio was 9:2. For the strain expressing the OnuE11 desaturase, no new compounds could be detected, indicating that this desaturase is most likely not well expressed or active in Y. lipolytica . Likewise, no activity was detected for the desaturases, EpoE11 and CpaE11, which were previously reported to produce E 11-14:acid in another yeast,Saccharomyces cerevisiae , albeit in minuscule amounts [29,31]. The strain expressing CroZ11 produced a mixture ofE /Z 11-14:acid with an excess of the Z isomer; however, the amounts of both isomers were lower compared to the strain expressing Lbo_PPTQ. The desaturase Hzead11 appeared to be a promiscuous ∆11 desaturase with a high preference towards 16:acid (Figure 1). Apart fromZ 11-16:acid, we also detected E /Z 11-14:acid and some other unsaturated fatty acids, which remain to be identified. (Figure S2). The Msextad11 desaturase-expressing strain producedE /Z 11-14:acid in ratio 1:1, where the amount of the E isomer was the highest among the tested desaturases.
In summary, Lbo_PPTQ resulted in the highest content and purity ofZ 11-14:acid and was therefore chosen for establishing the biosynthetic pathway towards ECB pheromone in Y. lipolytica .