Fatty acyl reductases (FARs) are involved in the biosynthesis of fatty alcohols that serve a range of biological roles. Insects typically harbor numerous FAR gene family members. While some FARs are involved in pheromone biosynthesis, the biological significance of the large number of FARs in insect genomes remains unclear.Using bumble bee (Bombini) FAR expression analysis and functional characterization, hymenopteran FAR gene tree reconstruction, and inspection of transposable elements (TEs) in the genomic environment of FARs, we uncovered a massive expansion of the FAR gene family in Hymenoptera, presumably facilitated by TEs. The expansion occurred in the common ancestor of bumble bees and stingless bees (Meliponini). We found that bumble bee FARs from the expanded FAR-A ortholog group contribute to the species-specific pheromone composition. Our results indicate that expansion and functional diversification of the FAR gene family played a key role in the evolution of pheromone communication in Hymenoptera.

There are more than one million described insect species. This species richness is reflected in the diversity of insect metabolic processes. In particular, biosynthesis of secondary metabolites, such as defensive compounds and chemical signals, encompasses an extraordinarily wide range of chemicals that are generally unparalleled among natural products from other organisms. Insect genomes, transcriptomes and proteomes thus offer a valuable resource for discovery of novel enzymes with potential for biotechnological applications. Here, we focus on fatty acid (FA) metabolism-related enzymes, notably the fatty acyl desaturases and fatty acyl reductases involved in the biosynthesis of FA-derived pheromones. Research on insect pheromone-biosynthetic enzymes, which exhibit diverse enzymatic properties, has the potential to broaden the understanding of enzyme specificity determinants and contribute to engineering of enzymes with desired properties for biotechnological production of FA derivatives. Additionally, the application of such pheromone-biosynthetic enzymes represents an environmentally friendly and economic alternative to the chemical synthesis of pheromones that are used in insect pest management strategies.

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Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18–26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L.

19The conserved fatty acyl reductase (FAR) family is involved in biosynthesis of fatty alcohols that 20 serve a range of biological roles. In moths, butterflies (Lepidoptera), and bees (Hymenoptera), 21FARs biosynthesize fatty alcohol pheromones participating in mate-finding strategies. Using a 22 combination of next-generation sequencing, analysis of transposable elements (TE) in the 23 genomic environment of FAR genes, and functional characterization of FARs from Bombus 24 lucorum, B. lapidarius, and B. terrestris, we uncovered a massive expansion of the FAR gene 25 family in Hymenoptera, presumably facilitated by TEs. Expansion occurred in the common 26 ancestor of bumblebees (Bombini) and stingless bees (Meliponini) after their divergence from the 27 honeybee lineage. We found that FARs from the expanded FAR-A orthology group contributed to 28 the species-specific male marking pheromone composition. Our results indicate that TE-mediated 29 expansion and functional diversification of the FAR gene family played a key role in the 30 evolution of pheromone communication in the crown group of Hymenoptera. 31 32 33 34 35 Abbreviations: MMP: male marking pheromone, FA: fatty acid, FAME: fatty acid methyl ester, 36 FAR: fatty acyl reductase, LG: labial gland, FB: fat body, TE: transposable element. 37 38 (Lepidoptera) are the most well-studied model of insect pheromone biosynthesis and have been 64 the subject of substantial research effort related to FARs. Variation in FAR enzymatic specificities 65 is a source of sex pheromone signal diversity among moths in the genus Ostrinia 19 and is also 66 responsible for the distinct pheromone composition in two reproductively isolated races of the 67European corn borer Ostrinia nubilalis 20 . Divergence in pheromone biosynthesis can potentially 68 install or strengthen reproductive barriers, ultimately leading to speciation 21 . However, the 69 biological significance of a large number of insect FAR paralogs remains unclear, as all FARs 70 implicated in moth and butterfly sex pheromone biosynthesis are restricted to a single clade, 71indicating that one FAR group was exclusively recruited for pheromone biosynthesis 20,22-24 . 72 While more than 20 FARs have been experimentally characterized from 23 moth and butterfly 73 (Lepidoptera) species 25 , FARs from other insect orders have received far less attention. Single 74 FAR genes have been isolated and experimentally characterized from Drosophila (Diptera) 14 , the 75 European honeybee (Hymenoptera) 26 and the scale insect Ericeus pela (Hemiptera) 27 . Our 76 limited knowledge about FAR function prevents us from drawing inferences about the biological 77 significance of the FAR gene family expansion in insects. 78 Bumblebees (Hymenoptera: Apidae) are a convenient experimental model to study 79 insect FAR evolution because the majority of bumblebee species produces fatty alcohols as 80 species-specific components of male marking pheromones (MMPs) 28 , which are presumed to be 81 biosynthesized by some of the numerous bumbl...

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One of the most interesting groups of fatty acid derivates is the group of conjugated fatty acids, from which the most researched are conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which are associated with countless health benefits. Sex pheromone mixture of some insect species, including tobacco horn-worm (Manduca sexta), are typical for the production of uncommon C16 long conjugated fatty acids with two and three conjugated double bonds, as opposed to C18 long CLA and CLNA. In this study, M. sexta desaturases MsexD2 and MsexD3 were expressed in multiple strains of Y. lipolytica with different genotypes. Experiments with supplementation of fatty acid methyl esters into the medium resulted in production of novel fatty acids. Using GCxGC-MS 20 new fatty acids with two or three double bonds were identified. Fatty acids with conjugated or isolated double bonds or combination of both were produced in trace amounts. Results of this study prove that Y. lipolytica is capable of synthesizing C16 conjugated fatty acids. Further genetic optimization of the Y. lipolytica genome and optimization of the fermentation process could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.