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. 2021 Jun;16(6):e2100004.
doi: 10.1002/biot.202100004. Epub 2021 Mar 18.

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

Karolis Petkevicius  1   2 , Eleni Koutsoumpeli  3 , Petri Christina Betsi  3 , Bao-Jian Ding  4 , Kanchana Rueksomtawin Kildegaard  2 , Hilbert Jensen  2 , Nora Mezo  2 , Andrea Mazziotta  2 , Anders Gabrielsson  2 , Christina Sinkwitz  2 , Bettina Lorantfy  2 , Carina Holkenbrink  2 , Christer Löfstedt  4 , Dimitris Raptopoulos  5 , Maria Konstantopoulou  3 , Irina Borodina  1   2
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Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica

Karolis Petkevicius et al. Biotechnol J. 2021 Jun.

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.

Keywords: Yarrowia lipolytica; fatty acyl-CoA desaturases; fatty acyl-CoA reductases; fatty alcohols; insect pheromones.

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References

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