No abstract

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations.1,2 Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits.3 Here we demonstrate a biotechnological method for the production of pheromones of economically important moth pests using engineered yeast cell factories. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a synthetic pheromone mixture. We further demonstrated the production of the main pheromone component of the fall armyworm Spodoptera frugiperda. Our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control by yeast fermentation.Significance statementAgriculture largely relies on insecticides and genetically modified crops for pest control, however alternative solutions are required due to emerging resistance, toxicity and regulatory issues, and consumer preferences. Mating disruption with sex pheromones that act by preventing insect reproduction is considered the most promising and scalable alternative to insecticides. This method is highly efficient and safe for human health and environment. The likelihood of insect resistance development is very low and can be handled by adjusting the pheromone composition. The high cost of chemically synthesized pheromones is the major barrier for the wider adoption of pheromones. A novel method based on yeast fermentation enables the production of insect sex pheromones as a lower cost from renewable feedstocks.

The formation of embryonic mineralized skeletal elements (spicules) in the sea urchin requires the participation of proteins, many of which may interact with one another and assist in the creation of an extracellular matrix wherein mineral formation takes place. To probe this, we created a sea urchin spicule recombinant model protein pair system wherein we tested the interactions between two major spicule proteins, SpSM50 and the glycoprotein, SpSM30B/C. Both proteins are strong hydrogelators that manipulate early and later events in mineral formation. We discovered that the anionic glycan moieties of SpSM30B/C are required for interaction with the SpSM50 protein and that these interactions are Ca(II)-independent. In addition, when these proteins form a complex, they create hybrid hydrogel particles that are physically distinct from their individual counterparts. Thus, glycan-mediated interactions play an important role in in vitro spicule protein assembly and most likely within the spicule itself.

In the nacre layer of the Pinctada fucata oyster shell there exists a multimember proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally defined combinatorial model consisting of two recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain (vWA)) and r-n16.3 (containing an EGF-like domain), whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca-mediated protein-protein interactions (∼4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp/Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization.

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.