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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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Constructs for co-expression of AseΔ11 and AseFAR in yeast. A) represents the generation of the construct Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT by homologous recombination through co-transformation in Saccharomyces cerevisiae of the AseFAR (yellow), which is flanked with short sequences (red) homologous to the PvuII-linearized pYEXCHT already carrying Ase∆11 (blue). B) represents how the two ORFs were fused into a single ORF by S. cerevisiae homologous recombination, generating the construct Ase∆11-FAR/CUP1p-pYEXCHT. The ORFs are flanked with a short sequence (red) homologous to the XhoI-linearized pYEXCHT on one side, as well as with a short homologous sequence interspacing the two ORFs (green) for the purpose to aid fusion into a chimeric ORF.
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Figure 1: Constructs for co-expression of AseΔ11 and AseFAR in yeast. A) represents the generation of the construct Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT by homologous recombination through co-transformation in Saccharomyces cerevisiae of the AseFAR (yellow), which is flanked with short sequences (red) homologous to the PvuII-linearized pYEXCHT already carrying Ase∆11 (blue). B) represents how the two ORFs were fused into a single ORF by S. cerevisiae homologous recombination, generating the construct Ase∆11-FAR/CUP1p-pYEXCHT. The ORFs are flanked with a short sequence (red) homologous to the XhoI-linearized pYEXCHT on one side, as well as with a short homologous sequence interspacing the two ORFs (green) for the purpose to aid fusion into a chimeric ORF.

Mentions: In this article, we report on the functional characterization of a FAD with Δ11-activity and a broad-acting pgFAR isolated from the Turnip moth, Agrotis segetum (Figure 1A-B) and investigate their heterologous co-expression by constructing a two-step pheromone biosynthetic pathway in yeast with the aim of producing (Z)-11-hexadecenol (Z11-16:OH, [24]), a common moth pheromone component, de novo. Our results constitute a proof of concept that it is indeed feasible to produce one of the most frequently used moth sex pheromone components in this way. With an increasing repertoire of characterized insect FADs and FARs at hand, this method has the potential to assist in the semi-synthetic production of various species-specific moth sex pheromone components.


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)

Constructs for co-expression of AseΔ11 and AseFAR in yeast. A) represents the generation of the construct Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT by homologous recombination through co-transformation in Saccharomyces cerevisiae of the AseFAR (yellow), which is flanked with short sequences (red) homologous to the PvuII-linearized pYEXCHT already carrying Ase∆11 (blue). B) represents how the two ORFs were fused into a single ORF by S. cerevisiae homologous recombination, generating the construct Ase∆11-FAR/CUP1p-pYEXCHT. The ORFs are flanked with a short sequence (red) homologous to the XhoI-linearized pYEXCHT on one side, as well as with a short homologous sequence interspacing the two ORFs (green) for the purpose to aid fusion into a chimeric ORF.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Constructs for co-expression of AseΔ11 and AseFAR in yeast. A) represents the generation of the construct Ase∆11/CUP1p-AseFAR/GAL1p-pYEXCHT by homologous recombination through co-transformation in Saccharomyces cerevisiae of the AseFAR (yellow), which is flanked with short sequences (red) homologous to the PvuII-linearized pYEXCHT already carrying Ase∆11 (blue). B) represents how the two ORFs were fused into a single ORF by S. cerevisiae homologous recombination, generating the construct Ase∆11-FAR/CUP1p-pYEXCHT. The ORFs are flanked with a short sequence (red) homologous to the XhoI-linearized pYEXCHT on one side, as well as with a short homologous sequence interspacing the two ORFs (green) for the purpose to aid fusion into a chimeric ORF.
Mentions: In this article, we report on the functional characterization of a FAD with Δ11-activity and a broad-acting pgFAR isolated from the Turnip moth, Agrotis segetum (Figure 1A-B) and investigate their heterologous co-expression by constructing a two-step pheromone biosynthetic pathway in yeast with the aim of producing (Z)-11-hexadecenol (Z11-16:OH, [24]), a common moth pheromone component, de novo. Our results constitute a proof of concept that it is indeed feasible to produce one of the most frequently used moth sex pheromone components in this way. With an increasing repertoire of characterized insect FADs and FARs at hand, this method has the potential to assist in the semi-synthetic production of various species-specific moth sex pheromone components.

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