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.
Bibliographical noteFunding Information:
Karolis Petkevicius acknowledges the funding from Innovationsfonden under grant agreement number 8053‐00179B. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 760798 (OLEFINE) and from the Bio‐Based Industries Joint Undertaking (BBI JU) under the European Union's Horizon 2020 research and innovation programe under grant agreement No: 886662 (PHERA). Irina Borodina and Christer Löfstedt acknowledge the financial support from the Novo Nordisk Foundation under grant agreements No. NNF15OC0016592 and No. NNF10CC1016517. Christer Löfstedt acknowledges funding from the Swedish Foundation for Strategic Research (No. RBP 14–0037, Oil Crops for the Future). The authors thank the Agricultural Research Service (NRRL, USA) for the yeast strain. The authors also thank Blanka Tóth for assistance with monitoring and sampling during fed‐batch fermentation.
© 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH
- Fatty acyl-CoA desaturases
- Fatty acyl-CoA reductases
- Fatty alcohols
- Insect pheromones
- Yarrowia lipolytica