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A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory.

Hagström Å, Wang HL, Liénard MA, Lassance JM, Johansson T, Löfstedt C - Microb. Cell Fact. (2013)

Bottom Line: We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum.A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH.This study is a first proof-of-principle that it is possible to "brew" biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pheromone Group, Department of Biology, Lund University, Lund, Sweden. asa.hagstrom@biol.lu.se.

ABSTRACT

Background: Moths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory.

Results: We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer's yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different species can be used as a molecular toolbox to produce pheromone components or pheromone component precursors of potential use for control of a variety of moths.

Conclusions: This study is a first proof-of-principle that it is possible to "brew" biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors. Substrates present in the yeast alone appear to be sufficient.

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Expression and co-expression of AseΔ11 and AseFAR in S. cerevisiae. A) Alcohol products from AseFAR expression (top) supplemented with Z11-16:ME, compared to vector only negative control (bottom). AseFAR produces the corresponding Z11-16:OH, as well as 12:OH, 14:OH, 16:OH, and Z9-16:OH, as a result from conversion of natural FA substrates present in yeast. None of these alcohols are found in the negative control. B) DMDS derivatization of Ase∆11-expressing yeast subjected to base methanolysis (top) compared to an empty vector control (bottom). The Ase∆11 chromatogram shows peaks corresponding to ∆11-12:DMDS, Z11-14:DMDS, E11-14:DMDS, and Z11-15:DMDS, which are not present in the negative control. Additionally, there is a substantially higher peak in AseΔ11 corresponding to Z11-16:DMDS (also see additional file 1). C) Base methanolysed yeast extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (top panel) as compared to a negative control (bottom panel) showing the presence of Z11-16:ME in the former but not the latter. D) Alcohol extracts from AseFAR-pYES2.1 (top panel) compared with Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (bottom panel). Both samples contain 12:OH, 14:OH, 16:OH, and Z9-16:OH, but only the latter contains Z11-16:OH.
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Figure 3: Expression and co-expression of AseΔ11 and AseFAR in S. cerevisiae. A) Alcohol products from AseFAR expression (top) supplemented with Z11-16:ME, compared to vector only negative control (bottom). AseFAR produces the corresponding Z11-16:OH, as well as 12:OH, 14:OH, 16:OH, and Z9-16:OH, as a result from conversion of natural FA substrates present in yeast. None of these alcohols are found in the negative control. B) DMDS derivatization of Ase∆11-expressing yeast subjected to base methanolysis (top) compared to an empty vector control (bottom). The Ase∆11 chromatogram shows peaks corresponding to ∆11-12:DMDS, Z11-14:DMDS, E11-14:DMDS, and Z11-15:DMDS, which are not present in the negative control. Additionally, there is a substantially higher peak in AseΔ11 corresponding to Z11-16:DMDS (also see additional file 1). C) Base methanolysed yeast extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (top panel) as compared to a negative control (bottom panel) showing the presence of Z11-16:ME in the former but not the latter. D) Alcohol extracts from AseFAR-pYES2.1 (top panel) compared with Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (bottom panel). Both samples contain 12:OH, 14:OH, 16:OH, and Z9-16:OH, but only the latter contains Z11-16:OH.

Mentions: When AseΔ11 was heterologously expressed in S. cerevisiae, the GC-MS analysis of base methanolysed cell extracts revealed the presence of an abundant peak corresponding to a hexadecenoic methyl ester with the characteristic ions m/z 268 and 236. The DMDS (dimethyl disulfide) derivative of this ester displayed the characteristic ions at m/z 245, 117 and  362, thus confirming a Δ11-double bond position. Further comparison of retention times with the reference compounds (E)-11-hexadecenoic acid methyl ester (E11-16:ME) and (Z)-11-hexadecenoic acid methyl ester (Z11-16:ME) confirmed a ¨Z¨ configuration of the double bond (Figure 3B and Additional file 1). AseΔ11-pYEXCHT produced significantly large amounts of (Z)-11-hexadecenoic acid (Z11-16:COOH), whereas the negative control (empty vector) produced much less of Z11-16:COOH, likely resulting from endogenous elongation of (Z)-9-tetradecenoic acid (Z9-14:COOH). In addition to Z11-16:COOH, the AseΔ11-pYEXCHT extract also contained (Δ)-11-dodecenoic acid (Δ11-12:COOH), (Z)-11-tetradecenoic acid (Z11-14:COOH), (E)-11-tetradecenoic acid (E11-14:COOH) and (Z)-11-pentadecenoic acid (Z11-15:COOH), as confirmed by the DMDS adducts of the methanolysis products. None of these acids were present in the negative control (Figure 3B). This Δ11-desaturase activity profile is a common pattern found in many moth species. Yet it is interesting that the Δ11-desaturase found in A. segetum shows no activity on 18:COOH but instead activities shifted towards C12-C16 acids. This distinguishes AseΔ11 from other identified and functionally characterized Δ11-desaturases that phylogenetically clustered into the same clade (Figure 2): its homolog found in Helicoverpa zea which is 16:COOH-specific [16], and possibly also from the homologs in Trichoplusia ni[27,28] and Spodoptera littoralis [29] that act on C14-C18 acids, as well as from an inactive copy from Mamestra brassicae [30]. This again exemplifies the biochemical diversity present amongst a small fraction of known moth pheromone biosynthetic Δ11-FADs, as compared to the conserved specificity and activity in Δ9-desaturase homologs, which are mostly metabolic.


A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory.

Hagström Å, Wang HL, Liénard MA, Lassance JM, Johansson T, Löfstedt C - Microb. Cell Fact. (2013)

Expression and co-expression of AseΔ11 and AseFAR in S. cerevisiae. A) Alcohol products from AseFAR expression (top) supplemented with Z11-16:ME, compared to vector only negative control (bottom). AseFAR produces the corresponding Z11-16:OH, as well as 12:OH, 14:OH, 16:OH, and Z9-16:OH, as a result from conversion of natural FA substrates present in yeast. None of these alcohols are found in the negative control. B) DMDS derivatization of Ase∆11-expressing yeast subjected to base methanolysis (top) compared to an empty vector control (bottom). The Ase∆11 chromatogram shows peaks corresponding to ∆11-12:DMDS, Z11-14:DMDS, E11-14:DMDS, and Z11-15:DMDS, which are not present in the negative control. Additionally, there is a substantially higher peak in AseΔ11 corresponding to Z11-16:DMDS (also see additional file 1). C) Base methanolysed yeast extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (top panel) as compared to a negative control (bottom panel) showing the presence of Z11-16:ME in the former but not the latter. D) Alcohol extracts from AseFAR-pYES2.1 (top panel) compared with Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (bottom panel). Both samples contain 12:OH, 14:OH, 16:OH, and Z9-16:OH, but only the latter contains Z11-16:OH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Expression and co-expression of AseΔ11 and AseFAR in S. cerevisiae. A) Alcohol products from AseFAR expression (top) supplemented with Z11-16:ME, compared to vector only negative control (bottom). AseFAR produces the corresponding Z11-16:OH, as well as 12:OH, 14:OH, 16:OH, and Z9-16:OH, as a result from conversion of natural FA substrates present in yeast. None of these alcohols are found in the negative control. B) DMDS derivatization of Ase∆11-expressing yeast subjected to base methanolysis (top) compared to an empty vector control (bottom). The Ase∆11 chromatogram shows peaks corresponding to ∆11-12:DMDS, Z11-14:DMDS, E11-14:DMDS, and Z11-15:DMDS, which are not present in the negative control. Additionally, there is a substantially higher peak in AseΔ11 corresponding to Z11-16:DMDS (also see additional file 1). C) Base methanolysed yeast extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (top panel) as compared to a negative control (bottom panel) showing the presence of Z11-16:ME in the former but not the latter. D) Alcohol extracts from AseFAR-pYES2.1 (top panel) compared with Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT (bottom panel). Both samples contain 12:OH, 14:OH, 16:OH, and Z9-16:OH, but only the latter contains Z11-16:OH.
Mentions: When AseΔ11 was heterologously expressed in S. cerevisiae, the GC-MS analysis of base methanolysed cell extracts revealed the presence of an abundant peak corresponding to a hexadecenoic methyl ester with the characteristic ions m/z 268 and 236. The DMDS (dimethyl disulfide) derivative of this ester displayed the characteristic ions at m/z 245, 117 and  362, thus confirming a Δ11-double bond position. Further comparison of retention times with the reference compounds (E)-11-hexadecenoic acid methyl ester (E11-16:ME) and (Z)-11-hexadecenoic acid methyl ester (Z11-16:ME) confirmed a ¨Z¨ configuration of the double bond (Figure 3B and Additional file 1). AseΔ11-pYEXCHT produced significantly large amounts of (Z)-11-hexadecenoic acid (Z11-16:COOH), whereas the negative control (empty vector) produced much less of Z11-16:COOH, likely resulting from endogenous elongation of (Z)-9-tetradecenoic acid (Z9-14:COOH). In addition to Z11-16:COOH, the AseΔ11-pYEXCHT extract also contained (Δ)-11-dodecenoic acid (Δ11-12:COOH), (Z)-11-tetradecenoic acid (Z11-14:COOH), (E)-11-tetradecenoic acid (E11-14:COOH) and (Z)-11-pentadecenoic acid (Z11-15:COOH), as confirmed by the DMDS adducts of the methanolysis products. None of these acids were present in the negative control (Figure 3B). This Δ11-desaturase activity profile is a common pattern found in many moth species. Yet it is interesting that the Δ11-desaturase found in A. segetum shows no activity on 18:COOH but instead activities shifted towards C12-C16 acids. This distinguishes AseΔ11 from other identified and functionally characterized Δ11-desaturases that phylogenetically clustered into the same clade (Figure 2): its homolog found in Helicoverpa zea which is 16:COOH-specific [16], and possibly also from the homologs in Trichoplusia ni[27,28] and Spodoptera littoralis [29] that act on C14-C18 acids, as well as from an inactive copy from Mamestra brassicae [30]. This again exemplifies the biochemical diversity present amongst a small fraction of known moth pheromone biosynthetic Δ11-FADs, as compared to the conserved specificity and activity in Δ9-desaturase homologs, which are mostly metabolic.

Bottom Line: We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum.A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH.This study is a first proof-of-principle that it is possible to "brew" biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pheromone Group, Department of Biology, Lund University, Lund, Sweden. asa.hagstrom@biol.lu.se.

ABSTRACT

Background: Moths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory.

Results: We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer's yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different species can be used as a molecular toolbox to produce pheromone components or pheromone component precursors of potential use for control of a variety of moths.

Conclusions: This study is a first proof-of-principle that it is possible to "brew" biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors. Substrates present in the yeast alone appear to be sufficient.

Show MeSH
Related in: MedlinePlus