Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica

Abstract

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)‐11‐tetradecenol (Z11‐14:OH), which can then be chemically acetylated to (Z)‐11‐tetradecenyl acetate (Z11‐14:OAc), the main pheromone component of the Z‐race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl‐CoA was obtained by introducing a point mutation into the α‐subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F). The intracellular accumulation of myristic acid increased 8.4‐fold. Next, fatty acyl‐CoA desaturases (FAD) and fatty acyl‐CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L‐1 Z11‐14:OH was reached in small‐scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1‐fold. The native FAS1 gene's overexpression led to a further 1.5‐fold titer increase, reaching 93.9 ± 11.7 mg L‐1 in small‐scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed‐batch mode, 188.1 ± 13.4 mg L‐1 of Z11‐14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11‐14:OAc. Electroantennogram experiments showed that males of the Z‐race of O. nubilalis were responsive to biologically‐derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.

Developments in Fatty Acid-Derived Insect Pheromone Production Using Engineered Yeasts

“…Several strategies, including preventing endogenous fatty alcohol degradation, inhibiting acyl-CoA degradation, reducing the flux toward storage lipids, and increasing the supply of tetradecanoyl-CoA precursor, have been applied to improve pheromones production; consequently, the engineered Y. lipolytica strains could produce 73.6 mg/L of Z9-C14:OH (15-fold increase in titer over the background strain) and 2.57 g/L of Z11-C16:OH ( Table 1 ; Holkenbrink et al, 2020 ). Introduction of a point mutation into the α-subunit of FAS ( FAS2 I 1220 F ), and overexpression of an optimal combination of a fatty acyl-CoA desaturase (FAD; Lbo_PPTQ) from Lobesia botrana , FAR (HarFAR) from H. armigera and the gene encoding native FAS1 led to a final Z11-14:OH titer of 188.1 mg/L in fed-batch fermentation ( Table 1 ; Petkevicius et al, 2021 ). In another study, expression of the gene encoding FAR, BlapFAR 4 from Bombus lapidarius or BlucFAR1 from Bombus lucorum in Y. lipolytica , led to the production of bumblebee pheromones consisting of long-chain fatty alcohols.…”

Biosynthesis of insect sex pheromone precursors via engineered β-oxidation in yeast

“…Metabolic engineering efforts in yeast previously allowed to obtain Saccharomyces cerevisiae and Yarrowia lipolytica strains capable of producing insect pheromones and their precursors such as ( Z )-9-tetradecenyl acetate ( Z 9-14:OAc), ( E / Z )-11-tetradecenol ( E / Z 11-14:OH), ( Z )-11-hexadecenol ( Z 11-16:OH), and ( Z )-11-hexadecenal ( Z 11-16:Ald). Hagström et al 2013b , Holkenbrink et al 2020 , Jiang et al 2021 , Petkevicius et al 2021 ). The plants Nicotiana benthamiana , N. tabacum , and Camelina sativa have been used for production of ∆11 C 14 and C 16 fatty acid derivatives as well (Ding et al 2014 , Mateos-Fernández et al 2021 , Nešněrová et al 2004 , Ortiz et al 2020 ).…”

“…Currently, chemical synthesis is the primary source of pheromones. However, in recent years, microbial and plant-based production has also been developed (Ding et al 2014 , Hagström et al 2013a , Holkenbrink et al 2020 , Jiang et al 2021 , Petkevicius et al 2021 , Xia et al 2020 ).…”

“…Additionally, a recent study in C. sativa demonstrated the production of a more challenging pheromone precursor of the codling moth Cydia pomonella -( E , E )-8,10-dodecadienoic acid (Xia et al 2021 ). Our previous studies showed that strategies related to decreased degradation of fatty acids/alcohols and improved acyl-CoA supply are beneficial for the biosynthesis of C 14 and C 16 pheromones (Holkenbrink et al 2020 , Petkevicius et al 2021 ).…”

Presentation_1.pdf

“…Several strategies, including preventing endogenous fatty alcohol degradation, inhibiting acyl-CoA degradation, reducing the flux toward storage lipids, and increasing the supply of tetradecanoyl-CoA precursor, have been applied to improve pheromones production; consequently, the engineered Y. lipolytica strains could produce 73.6 mg/L of Z9-C14:OH (15-fold increase in titer over the background strain) and 2.57 g/L of Z11-C16:OH (Table 1; Holkenbrink et al, 2020). Introduction of a point mutation into the α-subunit of FAS (FAS2 I 1220F ), and overexpression of an optimal combination of a fatty acyl-CoA desaturase (FAD; Lbo_PPTQ) from Lobesia botrana, FAR (HarFAR) from H. armigera and the gene encoding native FAS1 led to a final Z11-14:OH titer of 188.1 mg/L in fed-batch fermentation (Table 1; Petkevicius et al, 2021). In another study, expression of the gene encoding FAR, BlapFAR4 from Bombus lapidarius or BlucFAR1 from Bombus lucorum in Y. lipolytica, led to the production of bumblebee pheromones consisting of long-chain fatty alcohols.…”