The pheromone brewery
Pheromones are environmentally friendly alternatives to the use of traditional pesticides for control of insect pests and indeed synthetic pheromones are annually produced in huge amounts for this purpose. We would like to overcome waste problems inherent to synthetic pheromone production by designing and developing an innovative green chemistry alternative, i.e., the synthesis of pheromones in a cost-effective and environmentally friendly plant factory or in yeast cell factories.
The projects aim at proof of principle, the demonstration of the feasibility of the production of moth pheromones in leafs and in yeast by the concerted expression of a suite of biosynthetic enzymes. A longterm vision is to design tailor-made production of any moth pheromone component in these systems. Such semi-synthetic preparation of sex pheromones will be a novel and cost-effective way of producing moderate to large quantities of pheromones with high purity and a minimum of nonhazardous waste. Genetically modified plants may be used in inter-cropping as natural dispensers of insect pheromones. The proposed strategy is innovative, environmentally friendly, involves no phytosanitary risks, contains fundamental research challenges and has the potential to become an economically sound part of many IPM programs.
Insect pheromone biosynthesis has been a major area of research in our laboratory during the last 30 years. Since the late 1990s great progress has been made on the molecular analysis of pheromone biosynthesis. Fatty acid-derived pheromone components in insects, in particular in moths, are biosynthesized by the interaction of specialised enzymes with trivial fatty acids. Double bonds in different positions and with different geometry are introduced in the precursors by the action of various desaturases. The fatty acid intermediates are then reduced to fatty alcohols and the alcohols may be subsequently oxidized or acetylated to produce aldehydes and acetates respectively. The vast majority of moth pheromones consist of mixtures of this type of alcohol, aldehyde or acetate pheromone components.
In vivo expression systems have allowed the confirmation of the functional identities of many moth desaturase-encoding mRNAs and more recently reductases (FARs). We have a wide range of representatives of the desaturase multigene family at hand, which when expressed in yeast produce fatty acids of different chain-length with double bonds in different positions. Our available genetic constructs include also several functionally characterized fatty-acyl reductases. We have demonstrated that substitution of one amino can account for remarkable changes in specificity, which provides an opportunity to re-engineer reductases with tailor-made substrate specificity. The genes and techniques available to us thus constitute a potent tool box from which we can pick up elements to be used in the construction of genetically modified yeast and plants with an ability to produce insect pheromones.
Expression protocol and analysis of metabolic products by gas chromatography-mass spectrometry (GC-MS). (A) Ligation of the gene of interest into a recombinant vector (1), transformation into competent bacteria cells (E. coli), cell selection (antibiotics) multiplication (2). Checking of vector construction (gene presence and insertion in frame) by PCR (3a) and sequencing information (3b). (B) Transformation into a yeast strain (S. cerevisiae) and growth of yeast colonies containing either a gene of interest or a control (vector only) (4). Yeast culture (5): Pre-induction phase (5a) followed by induction of a specific promoter (e.g. Gal) that control gene expression (5b). (C) GC-MS) analyses from yeast total lipid extracts (6). Comparison of chromatograms obtained from control cells and cells expressing the introduced gene allow determination of the gene function(s) (7). Graphics: Marjorie Liénard.
- PlantLink. Sten Stymne and Per Hofvander, Department of Plant Breeding and Biotechnology, SLU, Alnarp
- Irina Borodina, DTU Biosustain, Techn. Univ. of Denmark, Hørsholm, Denmark
- Erik Hedenström, Organic Chemistry, Mid Sweden University, Sundsvall
- Jean-Marc Lassance, Harvard University, USA
- Marjorie Liénard, Harvard University, USA