Yeast engineering to express sex pheromone gland genes of the oriental fruit moth, Grapholita molesta

Abstract

Abstract Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at ∆ 9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at ∆ 9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.