Pheromones are utilized to a great extent in insects. Many of these pheromones are biosynthesized through a pathway involving fatty acids. This chapter will provide examples where the biosynthetic pathways of fatty acid-derived pheromones have been studied in detail. These include pheromones from Lepidoptera, Coleoptera, and Hymenoptera. Many species of Lepidoptera utilize fatty acids as precursors to pheromones with a functional group that include aldehydes, alcohols, and acetate esters. In addition, the biosynthesis of hydrocarbons will be briefly examined because many insects utilize hydrocarbons or modified hydrocarbons as pheromones.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

Fatty acyl reductases (FARs) are key enzymes that participate in sex pheromone biosynthesis by reducing fatty acids to fatty alcohols. Lepidoptera typically harbor numerous FAR gene family members. Although FAR genes are involved in the biosynthesis of sex pheromones in moths, the key FAR gene of Spodoptera litura remains unclear. In this work, we predicted 30 FAR genes from the S. litura genome and identified a domain duplication within gene SlitFAR3, which exhibited high and preferential expression in the sexually mature female pheromone glands (PGs) and a rhythmic expression pattern during the scotophase of sex pheromone production. The molecular docking of SlitFAR3, as predicted using a 3D model, revealed a co-factor NADPH binding cavity and 2 substrate binding cavities. Functional expression in yeast cells combined with comprehensive gas chromatography indicated that the SlitFAR3 gene could produce fatty alcohol products. This study is the first to focus on the special phenomenon of FAR domain duplication, which will advance our understanding of biosynthesis-related genes from the perspective of evolutionary biology.

Wax esters are high-value compounds used as feedstocks for the production of lubricants, pharmaceuticals, and cosmetics. Currently, they are produced mostly from fossil reserves using chemical synthesis, but this cannot meet increasing demand and has a negative environmental impact. Natural wax esters are also obtained from Simmondsia chinensis (jojoba) but comparably in very low amounts and expensively. Therefore, metabolic engineering of plants, especially of the seed storage lipid metabolism of oil crops, represents an attractive strategy for renewable, sustainable, and environmentally friendly production of wax esters tailored to industrial applications. Utilization of wax ester-synthesizing enzymes with defined specificities and modulation of the acyl-CoA pools by various genetic engineering approaches can lead to obtaining wax esters with desired compositions and properties. However, obtaining high amounts of wax esters is still challenging due to their negative impact on seed germination and yield. In this review, we describe recent progress in establishing non-food-plant platforms for wax ester production and discuss their advantages and limitations as well as future prospects.

Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management.
In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant.
Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies.

Wax esters are high-value compounds used as feedstocks for the production of lubricants, pharmaceuticals and cosmetics. Currently, they are produced mostly from fossil reserves using chemical synthesis, which cannot meet the increasing demand and have a negative environmental impact. Natural wax esters are also obtained from Simmondsia chinensis (jojoba) but comparably in very minor and expensive amounts. Therefore, metabolic engineering of plants, especially of the seed storage lipid metabolism of oil crops, represents an attractive strategy for renewable, sustainable and environmentally friendly production of wax esters tailored to industrial applications. Utilization of wax ester-synthesizing enzymes with defined specificities and modulation of the acyl-CoA pools via various genetic engineering approaches can lead to obtaining wax esters with desired compositions and properties. However, obtaining high amounts of wax esters is still challenging due to their negative impact on seed germination and yield. In this review, we describe the recent progress in establishing non-food plant platforms for wax ester production and discuss their advantages and limitations as well as future prospects.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.

BACKGROUND: Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.
RESULTS: For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed-1 of wax esters, containing 27-34 mol% oleyl oleate.
CONCLUSIONS: The overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.

Wax synthases are involved in the biosynthesis of wax esters, lipids with great industrial potential. Here, we heterologously expressed the native wax synthase MhWS2 from Marinobacter hydrocarbonoclasticus in Saccharomyces cerevisiae and performed comprehensive analysis of its substrate specificity. The enzyme displayed high wax synthase (but no diacylglycerol acyltransferase) activity both in vivo and in vitro. In the presence of exogenous fatty alcohol, wax esters accounted for more than 57% of total yeast lipids. In vitro, MhWS2 produced wax esters with most of the tested substrates, showing the highest activity with 14:0-, 18:1-, 18:0-, 12:0-, and 16:0-CoA together with saturated C10-C16 fatty alcohols. Co-expression with genes encoding fatty acyl reductases resulted in the accumulation of C26-C36 wax esters. Altogether, our results provide a detailed characterization of MhWS2 which should be useful in the development of strategies for producing wax esters in various expression systems.

The sympatric closely related species Helicoverpa armigera and Helicoverpa assulta use 97:3 and 7:93 of (Z)-11-hexadecenal and (Z)-9-hexadecenal, respectively, as their sex pheromone to find/locate correct sex mates. Moreover, (Z)-11-hexadecenyl alcohol and (Z)-9-hexadecenyl alcohol are more abundant in the pheromone gland of H. assulta than in that of H. armigera. To clarify the molecular basis of these differences, we sequenced the pheromone gland transcriptomes of the two species and compared the expression patterns of the candidate enzyme genes involved in the pheromone biosynthetic pathways by FPKM values and quantitative RT-PCR analysis. We found that the desaturase gene LPAQ expressed about 70 times higher in H. armigera than in H. assulta, whereas another desaturase gene NPVE expressed about 60 times higher in H. assulta than in H. armigera. We also observed significantly higher expression of the fatty acyl reductase (FAR) gene FAR1 and the aldehyde reductase (AR) gene AR3 in H. assulta than in H. armigera. Examination of the pheromone glands of the backcross offspring of their hybrids to H. assulta showed a positive linear correlation between the expression level of LPAQ and the amount of Z11-16:Ald and between the expression level of NPVE and the amount of Z9-16:Ald in the pheromone glands. Taken together, these data demonstrate that the expressional divergences of LPAQ and NPVE determine the opposite sex pheromone component ratios in the two species and the divergent expression of FAR1 and AR3 may account for the greater accumulation of alcohols in the pheromone gland of H. assulta.