Heterologous Expression of Methylketone Synthase1 and Methylketone Synthase2 Leads to Production of Methylketones and Myristic Acid in Transgenic Plants    

Abstract

Some plants produce methylketones as potent defense compounds against various insects. Wild tomato (Solanum habrochaites), a relative of the cultivated tomato (Solanum lycopersicum), synthesizes large amounts of 2-methylketones in its glandular trichomes, but cultivated tomato trichomes contain little or no methylketones. Two enzymes, Solanum habrochaites methylketone synthase1 (ShMKS1) and ShMKS2, are required to convert b-ketoacyl acyl-carrier protein intermediates of the fatty acid biosynthetic pathway to methylketones. ShMKS2 is a thioesterase that hydrolyzes b-ketoacyl acyl-carrier protein, and ShMKS1 is a decarboxylase that converts the resulting 3-ketoacids to 2-methylketones. We introduced ShMKS2 by itself or together with ShMKS1 to Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and cultivated tomato under the control of the 35S, Rubisco small subunit, and tomato trichome-specific promoters. Young tobacco and Arabidopsis plants expressing both genes under the control of 35S and Rubisco small subunit promoters produced methylketones in their leaves but had serious growth defects. As plants matured, they ceased to produce methylketones. Tobacco plants but not Arabidopsis or tomato plants expressing only ShMKS2 under the 35S promoter also synthesized methylketones, but at a lower rate. Transgenic cultivated tomato plants expressing ShMKS1 and ShMKS2 under trichome-specific promoters had slightly elevated levels of methylketone. Trace amounts of myristic acid were also detected in transgenic plants constitutively expressing ShMKS2 with or without ShMKS1. These results suggest that increases in methylketone production in plants will require the targeting of the pathway to self-contained structures in the plant and may also require increasing the flux of fatty acid biosynthesis.

Recent progress in secondary metabolism of plant glandular trichomes

“…Due to the potential of methylketone as a biofuel, the MKS2 gene was integrated into E. coli, and the best production was around 380 mg/l in a fatty acid-overproducing strain (Goh et al 2012). However, a similar strategy in plant systems did not generate satisfactory results, probably due to the limited availability of substrates (Yu and Pichersky 2014). This result also pointed out the much higher complexity of plant systems as compared to microbial systems in metabolic engineering.…”

Transgenic RXLR Effector PITG_15718.2 Suppresses Immunity and Reduces Vegetative Growth in Potato

“…Interestingly, among upregulated DEGs, PGSC0003DMG400003608 , encoding an abscisic acid 8’-hydroxylase, is reminiscent of a previous finding of negatively regulated plant growth because overexpression of the homologue of GhABAH was found to inhibit the growth in transgenic tomatoes and cotton [53]. Overexpression of ShMKS1 in Arabidopsis thaliana and Nicotiana tabacum also caused serious growth defects [54]. The upregulated DEG PGSC0003DMG400025912 is a homologue of ShMKS, which encodes a salicylic acid-binding protein and may negatively regulate plant growth.…”

Biosynthesis of the Nematode Attractant 2-Heptanone and Its Co-evolution Between the Pathogenic Bacterium Bacillus nematocida and Non-pathogenic Bacterium Bacillus subtilis

“…Wild tomatoes produce methylketones as repellants against its pests (Yu, 2013), in which two genes shMKSI and shMKSII are identified to be responsible for the production of methylketones during fatty acid biosynthesis (Yu et al, 2010). In this pathway, ShMKS2 hydrolyzes β-ketoacyl-A to release 3-ketoacids, and then ShMKS1 catalyzes the decarboxylation of 3-ketoacids to produce methylketones (Yu et al, 2010; Yu and Pichersky, 2014). So the shMKSI gene is epistatic to shMKSII in the pathway for methylketone biosynthesis.…”

“…Collectively, it is widely believed that odd-numbered methylketones are synthesized from their corresponding even-numbered fatty acids via abortive β-oxidation (Schulz and Dickschat, 2007). However, it was recently shown that in wild tomatoes ( Solanum habrochaites ) that could produce methylketones, the gene expressional levels involved in fatty acid synthesis were much higher than those of fatty acid beta oxidation pathway (Fridman et al, 2005; Yu and Pichersky, 2014). Furthermore, two methylketone synthetase genes shMKSI and shMKSII have been identified to catalyze the substrate β-ketoacyl-ACP (β-ketoacyl-acyl carrier protein), which was one of the intermediate products in fatty acid synthesis, into the products of methylketone (Fridman et al, 2005).…”