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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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Gas chromatography of oxidized yeast extracts. (A) When stimulated with the oxidized extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT the H. virescens male antenna responds to the pheromone components Z9-14:Al and Z11-16:Al, as well as to 14:Al, Z9-16:Al and to a lesser extent to 16:Al. (B) In the negative control there are no aldehydes and no antennal responses to the oxidized yeast extract at corresponding retention times (dashed lines), or at any other elution time. Flame ionization detector (FID) response is marked in black, while electroantennographic detector (EAD) response using a male H. virescens antenna is marked in blue. Repeated experiments produced the same EAD response pattern.
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Figure 4: Gas chromatography of oxidized yeast extracts. (A) When stimulated with the oxidized extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT the H. virescens male antenna responds to the pheromone components Z9-14:Al and Z11-16:Al, as well as to 14:Al, Z9-16:Al and to a lesser extent to 16:Al. (B) In the negative control there are no aldehydes and no antennal responses to the oxidized yeast extract at corresponding retention times (dashed lines), or at any other elution time. Flame ionization detector (FID) response is marked in black, while electroantennographic detector (EAD) response using a male H. virescens antenna is marked in blue. Repeated experiments produced the same EAD response pattern.

Mentions: To validate the biological activity of the oxidized yeast extract and to support the idea that genes from one species can be used to produce components for other species as well, the Tobacco budworm, Heliothis virescens was selected as an experimental insect. There is a high structural similarity between the sex pheromone of this species, which mainly consists of (Z)-11-hexadecenal (Z11-16:Al) and (Z)-9-tetradecenal (Z9-14:Al) with traces of other aldehydes (Additional file 4), and the oxidized yeast extract, i.e., the aldehydes resulting from oxidation of the alcohols (Table 1). When H. virescens male antennae were subjected to a synthetically oxidized yeast extract resulting from the co-expression of AseΔ11 and AseFAR, there were strong responses to the major pheromone component Z11-16:Al, and the minor pheromone component Z9-14:Al (produced in minor amounts), as well as to 14:Al, 16:Al, and Z9-16:Al (Figure 4A). This was in agreement with our expectations and proved the electrophysiological activity of the oxidized yeast co-expression products, at the same time demonstrating the absence of side-products with potentially interfering electrophysiological activity. The oxidized extract from the negative control did not elicit any antennal response (Figure 4B).


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)

Gas chromatography of oxidized yeast extracts. (A) When stimulated with the oxidized extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT the H. virescens male antenna responds to the pheromone components Z9-14:Al and Z11-16:Al, as well as to 14:Al, Z9-16:Al and to a lesser extent to 16:Al. (B) In the negative control there are no aldehydes and no antennal responses to the oxidized yeast extract at corresponding retention times (dashed lines), or at any other elution time. Flame ionization detector (FID) response is marked in black, while electroantennographic detector (EAD) response using a male H. virescens antenna is marked in blue. Repeated experiments produced the same EAD response pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126085&req=5

Figure 4: Gas chromatography of oxidized yeast extracts. (A) When stimulated with the oxidized extract from Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT the H. virescens male antenna responds to the pheromone components Z9-14:Al and Z11-16:Al, as well as to 14:Al, Z9-16:Al and to a lesser extent to 16:Al. (B) In the negative control there are no aldehydes and no antennal responses to the oxidized yeast extract at corresponding retention times (dashed lines), or at any other elution time. Flame ionization detector (FID) response is marked in black, while electroantennographic detector (EAD) response using a male H. virescens antenna is marked in blue. Repeated experiments produced the same EAD response pattern.
Mentions: To validate the biological activity of the oxidized yeast extract and to support the idea that genes from one species can be used to produce components for other species as well, the Tobacco budworm, Heliothis virescens was selected as an experimental insect. There is a high structural similarity between the sex pheromone of this species, which mainly consists of (Z)-11-hexadecenal (Z11-16:Al) and (Z)-9-tetradecenal (Z9-14:Al) with traces of other aldehydes (Additional file 4), and the oxidized yeast extract, i.e., the aldehydes resulting from oxidation of the alcohols (Table 1). When H. virescens male antennae were subjected to a synthetically oxidized yeast extract resulting from the co-expression of AseΔ11 and AseFAR, there were strong responses to the major pheromone component Z11-16:Al, and the minor pheromone component Z9-14:Al (produced in minor amounts), as well as to 14:Al, 16:Al, and Z9-16:Al (Figure 4A). This was in agreement with our expectations and proved the electrophysiological activity of the oxidized yeast co-expression products, at the same time demonstrating the absence of side-products with potentially interfering electrophysiological activity. The oxidized extract from the negative control did not elicit any antennal response (Figure 4B).

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