RESUMEN
The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway.
Asunto(s)
Vías Biosintéticas , Cucurbitaceae/crecimiento & desarrollo , Proteínas de Plantas/genética , Triterpenos/metabolismo , Cucurbitaceae/genética , Cucurbitaceae/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ADN/métodos , Escualeno-Monooxigenasa/química , Escualeno-Monooxigenasa/genética , Escualeno-Monooxigenasa/metabolismoRESUMEN
Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon.
Asunto(s)
Citrullus/metabolismo , Cucurbitacinas/biosíntesis , Aromatizantes/metabolismo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Citrullus/química , Citrullus/enzimología , Citrullus/genética , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Alineación de Secuencia , GustoRESUMEN
Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.
Asunto(s)
Cucumis melo/genética , Cucumis sativus/genética , Frutas/química , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Ácido Cítrico/análisis , Cucumis melo/química , Cucumis sativus/química , Frutas/genética , Concentración de Iones de Hidrógeno , Solanum lycopersicum/química , Malatos/análisisRESUMEN
A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Secuencias Repetidas en Tándem/genética , Secuencia de Aminoácidos , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Secuencia de Bases , Clonación Molecular , Endotoxinas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hemolisinas/química , Datos de Secuencia MolecularRESUMEN
The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a beta-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.