Fatty Acyl Reductases and Fatty Acyl Desaturases. Deciphering the biosynthesis of species-specific moth female sex pheromones from common fatty acids

Sammanfattning: Moths (Lepidoptera) are dependent on female produced pheromones, chemical signals, for attracting a mate. Pheromones are most commonly made up by saturated and/or unsaturated fatty acid derived alcohols, acetates, and/or aldehydes that are produced by a set of enzymes in the female pheromone gland. The enzymes involved are fatty acyl desaturases, pheromone gland fatty acyl reductases (pgFAR), β-oxidases, acetyl transferases, and alcohol oxidases. Among these, the desaturases and the pgFARs are the only genes have been identified and characterized. The desaturases introduce a double bond into the fatty acyl carbon chain, and the pgFARs catalyze the reduction from a fatty acyl CoA precursor to the corresponding alcohol. I have worked with these two enzymes, focusing on understanding their role in the biosynthetic pathway of moths and to understand how they shape a set of rather common fatty acyl precursors to a highly species specific pheromone. A single FAR-like gene, FARII, was found expressed in the pheromone gland only in three Yponomeutids, in contrast to other potential FARs that were expressed in the fat body as well. A functional assay revealed a broad activity of FARII on substrates between C12 and C16, which included all the pheromone precursors in the three species. A phylogenetic analysis of different FARs showed that all pgFARs belonged to a Lepidoptera-specific clade. The pgFARs from four Heliothinae species were then identified and functionally characterized. The enzymes reduced a broad set of precursors between C8 and C16. A comparison between the pheromone gland precursor ratio and pheromone component ratio indicated that there was a modulating step occurring between these. Adding three of the most common fatty acid precursors in defined ratios to the enzymes, elucidated that the pgFARs had a hierarchical selectivity, preferring Z9-14:ME over Z11-16:ME that in turn was preferred over Z9-16:ME. This confirmed that pgFAR has a shaping role in the pheromone biosynthesis in these species. A new phylogenetic tree, also including other arthropod and mammalian homologs known to perform similar reductive activities, again supported the pgFAR clade. Then the subcellular localization of pgFAR from Heliothis virescens (HvFAR) was studied by constructing fusion proteins between HvFAR and Green Fluorescent Protein (GFP) and expressing it in yeast. The C-terminally tagged protein localized to the endoplasmic reticulum (ER) where it retained its catalytic properties, as a contrast to the N-terminally tagged protein that was expressed in the cytoplasm with no enzymatic activity present. This indicates that the N-terminal is important for both subcellular localization and enzyme functionality, which was also supported by a series of truncations. HvFAR topology was investigated with a Fluorescent Protease Protection (FPP) assay, which showed that the C-terminal is located on the cytoplasmic side of the ER membrane. Together with a series in silico topology predictions, this led to a model of the pgFARs where the active site of the enzyme is placed on the cytoplasmic side of the ER membrane. Since this is also the case for the desaturase, this suggests that the ER is an important site in the moth pheromone biosynthesis. A pgFAR and a desaturase from the turnip moth, Agrotis segetum, were then functionally characterized. Two types of constructs were made for co-expression of both enzymes in yeast, and both methods successfully produced Z11-16:OH, which neither the yeast nor each enzyme alone can produce. Oxidized yeast extracts used on male H. virescens antennae by gas chromatography with electroantennographic detection (GC-EAD) verified biological activity of the products. There was no response on any other yeast component, which supports the use of yeast as a cell factory. Finally, two orthologous desaturases from the tortricids Ctenopseustis obliquana and C. herana were studied. Both were found in each species’ pheromone gland transcriptome and shared 97% sequence identity, but were quite different from other known moth desaturases sharing only 57% with the closest homolog from a butterfly. A phylogenetic analysis confirmed that the Ctenopseustis spp. desaturases clustered in a group of their own, which was more close to desaturases of unknown function in other insects rather than those involved in moth pheromone biosynthesis. A functional assay revealed that the desaturase was active on and selective on myristic acid, producing Z5-14:ME, a pheromone intermediate in both C. obliquana and C. herana, which confirmed the enzyme’s involvement in the pheromone biosynthesis. These results have together demonstrated the important interplay between the enzymes and the precursor ratios as well as between the enzymes themselves. The results have also elucidated the diversity of pgFARs and desaturases in Lepidoptera, and this knowledge has in turn been used to produce pheromone components in yeast.