Bioproduction of (Z,E)-9,12-tetradecadienyl acetate (ZETA), the major pheromone component of Plodia, Ephestia, and Spodoptera species in yeast.
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ABSTRACT: Background
(Z,E)-9,12-tetradecadienyl acetate (ZETA, Z9,E12-14:OAc) is a major sex pheromone component for many stored-product moth species. This pheromone is used worldwide for mating disruption, detection, monitoring, and mass trapping in raw and processed food storage facilities. In this study, we demonstrate the biological production of ZETA pheromone by engineered yeast Saccharomyces cerevisiae.Results
We mined the pheromone gland transcriptome data of the almond moth, Ephestia (Cadra) cautella (Walker), to trace a novel E12 fatty acyl desaturase and expressed candidates heterologously in yeast and Sf9 systems. Furthermore, we demonstrated a tailor-made ZETA pheromone bioproduction in yeast through metabolic engineering using this E12 desaturase, in combination with three genes from various sources coding for a Z9 desaturase, a fatty acyl reductase, and an acetyltransferase, respectively. Electrophysiological assays (gas chromatography coupled to an electroantennographic detector) proved that the transgenic yeast-produced ZETA pheromone component elicits distinct antennal responses.Conclusion
The reconstructed biosynthetic pathway in yeast efficiently produces ZETA pheromone, leaves an undetectable level of biosynthetic intermediates, and paves the way for the economically competitive high-demand ZETA pheromone's bioproduction technology for high-value storage pest control.
SUBMITTER: Ding BJ
PROVIDER: S-EPMC9300079 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe fall armyworm Spodoptera frugiperda (Lepidoptera; Noctuidae) consists of two distinct strains with different host plant preferences for corn and rice. To assess whether pheromonal-mediated behavioral isolation accompanies the habitat isolation on different host plants, we compared the sex pheromone composition among females of the two strains. Pheromone glands were extracted with or without injection of pheromone biosynthesis activating neuropeptide (PBAN). To assess the mode of inheritance of this variation, we also analyzed the pheromone composition of F1 hybrid females.ResultsRelative to intra-strain variation, the pheromone composition of the two strains differed significantly. Corn strain females contained significantly more of the second most abundant pheromone compound Z11-16:Ac (m), and significantly less of most other compounds, than rice strain females. When females were injected with PBAN before their glands were extracted, the differences between the strains were less pronounced but still statistically significant. The pheromone composition of hybrid females showed a maternal inheritance of the major component Z9-14:Ac (M) as well as of Z11-16:Ac (m). Most other compounds showed an inheritance indicating genetic dominance of the corn strain. The within-strain phenotypic correlations among the various components were consistent with their hypothesized biosynthetic pathway, and between-strain differences in the correlation structure suggested candidate genes that may explain the pheromone differences between the two strains. These include Delta9- and Delta11 desaturases, and possibly also a Delta7-desaturase, although the latter has not been identified in insects so far.ConclusionThe two host strains of S. frugiperda produce systematically differing female sex pheromone blends. Previously-documented geographic variation in the sexual communication of this species did not take strain identity into account, and thus may be partly explained by different strain occurrence in different regions. The finding of pheromone differences reinforces the possibility of incipient reproductive isolation among these strains, previously shown to differ in the timing of nocturnal mating activity and host plant use. Finding the genetic basis of the pheromone differences, as well as these other biological traits, will help to elucidate the role of premating isolation in the continuing differentiation of these two strains that may eventually lead to speciation.
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Project description:The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.
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Project description:Cellular processes are subject to variability, or noise, yet mechanisms that promote cell-to-cell uniformity are poorly understood. We have identified such a role for Dig1, a redundant (with Dig2) MAPK-responsive inhibitor of the S. cerevisiae mating pathway transcription factor Ste12. Cells lacking Dig1, but not Dig2, exhibited increased variability in outputs of the mating pathway. dig1∆ mutants also displayed a Ste12-dependent defect in growth in the absence of mating pheromone and a quantitative defect in the process of mating, itself. In cells expressing two reporter genes driven by the same promoter, both intrinsic/uncorrelated and extrinsic/correlated noise were found to increase. Remarkably, the extrinsic noise phenotype in cells lacking Dig1 correlates with the aggregation of target genes: we observed subnuclear foci of Ste12 specifically in dig1∆ cells and, using a newly-developed method to immunoprecipitate a single locus from crosslinked chromatin, we found that Dig1 inhibits long-range interactions between Ste12 target genes in vivo. Dig1 may shield binding surfaces on Ste12 whose unmasking leads to inappropriate gene associations and increased gene expression noise. These studies reveal how investigations of variability modulation mechanisms can yield unexpected biological insights.
2010-12-15 | GSE17583 | GEO
Project description:BackgroundInsects use pheromones, chemical signals that underlie all animal behaviors, for communication and for attracting mates. Synthetic pheromones are widely used in pest control strategies because they are environmentally safe. The production of insect pheromones in transgenic plants, which could be more economical and effective in producing isomerically pure compounds, has recently been successfully demonstrated. This research requires information regarding the pheromone biosynthetic pathways and the characterization of pheromone biosynthetic enzymes (PBEs). We used Illumina sequencing to characterize the pheromone gland (PG) transcriptome of the Pyralid moth, Ephestia cautella, a destructive storage pest, to reveal putative candidate genes involved in pheromone biosynthesis, release, transport and degradation.ResultsWe isolated the E. cautella pheromone compound as (Z,E)-9,12-tetradecadienyl acetate, and the major pheromone precursors 16:acyl, 14:acyl, E14-16:acyl, E12-14:acyl and Z9,E12-14:acyl. Based on the abundance of precursors, two possible pheromone biosynthetic pathways are proposed. Both pathways initiate from C16:acyl-CoA, with one involving ∆14 and ∆9 desaturation to generate Z9,E12-14:acyl, and the other involving the chain shortening of C16:acyl-CoA to C14:acyl-CoA, followed by ∆12 and ∆9 desaturation to generate Z9,E12-14:acyl-CoA. Then, a final reduction and acetylation generates Z9,E12-14:OAc. Illumina sequencing yielded 83,792 transcripts, and we obtained a PG transcriptome of ~49.5 Mb. A total of 191 PBE transcripts, which included pheromone biosynthesis activating neuropeptides, fatty acid transport proteins, acetyl-CoA carboxylases, fatty acid synthases, desaturases, β-oxidation enzymes, fatty acyl-CoA reductases (FARs) and fatty acetyltransferases (FATs), were selected from the dataset. A comparison of the E. cautella transcriptome data with three other Lepidoptera PG datasets revealed that 45% of the sequences were shared. Phylogenetic trees were constructed for desaturases, FARs and FATs, and transcripts that clustered with the ∆14, ∆12 and ∆9 desaturases, PG-specific FARs and potential candidate FATs, respectively, were identified. Transcripts encoding putative pheromone degrading enzymes, and candidate pheromone carrier and receptor proteins expressed in the E. cautella PG, were also identified.ConclusionsOur study provides important background information on the enzymes involved in pheromone biosynthesis. This information will be useful for the in vitro production of E. cautella sex pheromones and may provide potential targets for disrupting the pheromone-based communication system of E. cautella to prevent infestations.
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Project description:Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.
| S-EPMC2140177 | biostudies-literature
Project description:Cellular processes are subject to variability, or noise, yet mechanisms that promote cell-to-cell uniformity are poorly understood. We have identified such a role for Dig1, a redundant (with Dig2) MAPK-responsive inhibitor of the S. cerevisiae mating pathway transcription factor Ste12. Cells lacking Dig1, but not Dig2, exhibited increased variability in outputs of the mating pathway. dig1∆ mutants also displayed a Ste12-dependent defect in growth in the absence of mating pheromone and a quantitative defect in the process of mating, itself. In cells expressing two reporter genes driven by the same promoter, both intrinsic/uncorrelated and extrinsic/correlated noise were found to increase. Remarkably, the extrinsic noise phenotype in cells lacking Dig1 correlates with the aggregation of target genes: we observed subnuclear foci of Ste12 specifically in dig1∆ cells and, using a newly-developed method to immunoprecipitate a single locus from crosslinked chromatin, we found that Dig1 inhibits long-range interactions between Ste12 target genes in vivo. Dig1 may shield binding surfaces on Ste12 whose unmasking leads to inappropriate gene associations and increased gene expression noise. These studies reveal how investigations of variability modulation mechanisms can yield unexpected biological insights. The FUS1 locus is tagged with an array of lac operators. Wild type and dig1∆ cells containing this tagged FUS1 locus and expressing a mCherry-LacI were grown up for ChIP-chip. The FUS1 locus was selectively immunoprecipitated using an anti-DsRed antibody.
2010-12-15 | E-GEOD-17583 | biostudies-arrayexpress