Orientin and vitexin production by a one-pot enzymatic cascade of a glycosyltransferase and sucrose synthase.

Orientin and vitexin, important components of bamboo-leaf extracts, are C-glycosylflavones which exhibit a number of interesting biological properties. In this work, we developed an efficient biocatalytic cascade for orientin and vitexin production consisting of Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). In order to relieve the bottleneck of the biocatalytic cascade, the biocatalytic efficiency, reaction condition compatibilities and the ratio of the enzymes were determined. We found that the specific activity of TcCGT was significantly influenced by enzyme dose and Triton X-100 or Tween 20 (0.2%). Co-culture of BL21-TcCGT-Co and BL21-GmSUS-Co affected the catalytic efficiency of TcCGT and GmSUS, and the maximum orientin production rate reached 47 µM/min at the inoculation ratio of 9:1. The optimal pH and temperature for the biocatalytic cascade were pH 7.5 and 30 °C, respectively. Moreover, the high dose of the enzymes can improve the tolerance of biocatalytic cascade to substrate inhibition in the one-pot reaction. By using a fed-batch strategy, maximal titers of orientin and vitexin reached 7090 mg/L with a corresponding molar conversion of 98.7% and 5050 mg/L with a corresponding molar conversion of 97.3%, respectively, which is the highest titer reported to date. Therefore, the method described herein for efficient production of orientin and vitexin by modulating catalytic efficiencies of enzymes can be widely used for the C-glycosylation of flavonoids.

Molecular and Structural Characterization of a Promiscuous C-Glycosyltransferase from Trollius chinensis.

Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 Šresolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.

An engineered oilseed crop produces oil enriched in two very long chain polyunsaturated fatty acids with potential health-promoting properties.

Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata showed that the transgenic plants produced the level of DDA and DTA at approximately 30% of the total fatty acids in seeds, and the amount of the two fatty acids remained stable over four generations. The oilseed crop producing a high and sustained level of DDA and DTA provides an opportunity for high value agricultural products for nutritional and medical uses.

Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from ß-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the ß-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of ß-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida.

Comparative analysis of benzoxazinoid biosynthesis in monocots and dicots: independent recruitment of stabilization and activation functions.

Benzoxazinoids represent preformed protective and allelophatic compounds that are found in a multitude of species of the family Poaceae (Gramineae) and occur sporadically in single species of phylogenetically unrelated dicots. Stabilization by glucosylation and activation by hydrolysis is essential for the function of these plant defense compounds. We isolated and functionally characterized from the dicot larkspur (Consolida orientalis) the benzoxazinoid-specific UDP-glucosyltransferase and ß-glucosidase that catalyze the enzymatic functions required to avoid autotoxicity and allow activation upon challenge by herbivore and pathogen attack. A phylogenetic comparison of these enzymes with their counterparts in the grasses indicates convergent evolution by repeated recruitment from homologous but not orthologous genes. The data reveal a great evolutionary flexibility in recruitment of these essential functions of secondary plant metabolism.

Genetic diversity in Delphinium staphisagria (Ranunculaceae), a rare Mediterranean dysploid larkspur with medicinal uses.

Delphinium staphisagria is an endemic annual or biennial herb from the Mediterranean Basin, widely distributed in isolated populations of variable size. We evaluated the allozyme diversity of 31 populations along its distribution range via starch gel electrophoresis, assaying 12 enzyme systems and scoring 17 loci. The low levels of genetic variability detected (A = 11.8, A(p) = 1.6, H(o) = 0.026, H(e) = 0.057), are discussed in relation to the life-history traits of the species, such as short life-span, selfing or gravity seed dispersion. Other factors influencing genetic diversity, such as evolutionary history and spreading are also considered. Due to its historical medicinal uses, this plant has probably become widespread in the Mediterranean area. Human-mediated distribution could have promoted few migrant genotypes, recent founder events and long distance dispersal. These events would explain the genetic homogeneity found within and among populations, as well as the absence of a clear biogeographic structure. The limited genetic variability, the high genetic similarity among populations and the dysploidy of this species make it worthy of conservation. Management strategies are proposed mainly to preserve its genetic pool.