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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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Phylogenetic analysis of AseΔ11 and AseFAR. A) Shows a Fatty-Acyl Desaturase (FAD) phylogenetic tree, in which Ase∆11 (pink) clusters with moth ∆11-desaturases in a subgroup of ∆11 noctuid homologs (grey green). B) Shows a Fatty-Acyl Reductase (FAR) phylogenetic tree, where the AseFAR clusters in a well-supported group (purple) of lepidopteran pgFARs (marked with triangles). Species abbreviations can be found in Additional file 5.
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Figure 2: Phylogenetic analysis of AseΔ11 and AseFAR. A) Shows a Fatty-Acyl Desaturase (FAD) phylogenetic tree, in which Ase∆11 (pink) clusters with moth ∆11-desaturases in a subgroup of ∆11 noctuid homologs (grey green). B) Shows a Fatty-Acyl Reductase (FAR) phylogenetic tree, where the AseFAR clusters in a well-supported group (purple) of lepidopteran pgFARs (marked with triangles). Species abbreviations can be found in Additional file 5.

Mentions: DNA sequencing of a pheromone gland EST clone (Acc. No ES583599, [25]) yielded a full-length AseΔ11 ORF of 1011 nt corresponding to a 336 aa protein, which clustered within a group of noctuid Δ11-desaturases in our phylogenetic analysis (Figure 2). The reconstructed phylogeny forms a catalogue of an enormous diversity of moth FADs, which are able to perform enzymatic reaction activities not known from other species. The ∆11-subfamily contains rapidly evolving genes with various activities, including not only Δ11 but for instance also Δ10, Δ6 and bifunctional enzymes [26-28]. Other non-lepidopteran, arthropod desaturases and vertebrate desaturases function as common metabolic Δ9-desaturases.


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)

Phylogenetic analysis of AseΔ11 and AseFAR. A) Shows a Fatty-Acyl Desaturase (FAD) phylogenetic tree, in which Ase∆11 (pink) clusters with moth ∆11-desaturases in a subgroup of ∆11 noctuid homologs (grey green). B) Shows a Fatty-Acyl Reductase (FAR) phylogenetic tree, where the AseFAR clusters in a well-supported group (purple) of lepidopteran pgFARs (marked with triangles). Species abbreviations can be found in Additional file 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Phylogenetic analysis of AseΔ11 and AseFAR. A) Shows a Fatty-Acyl Desaturase (FAD) phylogenetic tree, in which Ase∆11 (pink) clusters with moth ∆11-desaturases in a subgroup of ∆11 noctuid homologs (grey green). B) Shows a Fatty-Acyl Reductase (FAR) phylogenetic tree, where the AseFAR clusters in a well-supported group (purple) of lepidopteran pgFARs (marked with triangles). Species abbreviations can be found in Additional file 5.
Mentions: DNA sequencing of a pheromone gland EST clone (Acc. No ES583599, [25]) yielded a full-length AseΔ11 ORF of 1011 nt corresponding to a 336 aa protein, which clustered within a group of noctuid Δ11-desaturases in our phylogenetic analysis (Figure 2). The reconstructed phylogeny forms a catalogue of an enormous diversity of moth FADs, which are able to perform enzymatic reaction activities not known from other species. The ∆11-subfamily contains rapidly evolving genes with various activities, including not only Δ11 but for instance also Δ10, Δ6 and bifunctional enzymes [26-28]. Other non-lepidopteran, arthropod desaturases and vertebrate desaturases function as common metabolic Δ9-desaturases.

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