Structural modeling of two plant UDP-dependent sugar-sugar glycosyltransferases reveals a conserved glutamic acid residue that is a hallmark for sugar acceptor recognition.

Glycosylation is one of the common modifications of plant metabolites, playing a major role in the chemical/biological diversity of a wide range of compounds. Plant metabolite glycosylation is catalyzed almost exclusively by glycosyltransferases, mainly by Uridine-diphosphate dependent Glycosyltransferases (UGTs). Several X-ray structures have been determined for primary glycosyltransferases, however, little is known regarding structure-function aspects of sugar-sugar/branch-forming O-linked UGTs (SBGTs) that catalyze the transfer of a sugar from the UDP-sugar donor to an acceptor sugar moiety of a previously glycosylated metabolite substrate. In this study we developed novel insights into the structural basis for SBGT catalytic activity by modelling the 3d-structures of two enzymes; a rhamnosyl-transferase Cs1,6RhaT - that catalyzes rhamnosylation of flavonoid-3-glucosides and flavonoid-7-glucosides and a UGT94D1 - that catalyzes glucosylation of (+)-Sesaminol 2-O-ß-d-glucoside at the C6 of the primary sugar moiety. Based on these structural models and docking studies a glutamate (E290 or E268 in Cs1,6RhaT or UGT94D1, respectively) and a tryptophan (W28 or W15 in Cs1,6RhaT or UGT94D1, respectively) appear to interact with the sugar acceptor and are suggested to be important for the recognition of the sugar-moiety of the acceptor-substrate. Functional analysis of substitution mutants for the glutamate and tryptophan residues in Cs1,6RhaT further support their role in determining sugar-sugar/branch-forming GT specificity. Phylogenetic analysis of the UGT family in plants demonstrates that the glutamic-acid residue is a hallmark of SBGTs that is entirely absent from the corresponding position in primary UGTs.

The molecular and enzymatic basis of bitter/non-bitter flavor of citrus fruit: evolution of branch-forming rhamnosyltransferases under domestication.

Domestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched-chain flavanone glycosides, the predominant flavonoids in citrus. The flavor-determining biosynthetic step is catalyzed by two branch-forming rhamnosyltransferases that utilize flavanone-7-O-glucose as substrate. The 1,2-rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone-7-O-neohesperidosides whereas the 1,6-rhamnosytransferase leads to the tastelessflavanone-7-O-rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6-rhamnosyltransferase-encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non-bitter citrus species. Cs1,6RhaT was found to be a substrate-promiscuous enzyme catalyzing branched-chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch-forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branch-forming rhamnosyltransferases underlie the development of bitter or non-bitter species/varieties under domestication.

Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC.

Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.

Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus.

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.

Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene.

Citrus fruits possess unique aromas rarely found in other fruit species. While fruit flavor is composed of complex combinations of soluble and volatile compounds, several low-abundance sesquiterpenes, such as valencene, nootkatone, alpha-sinensal, and beta-sinensal, stand out in citrus as important flavor and aroma compounds. The profile of terpenoid volatiles in various citrus species and their importance as aroma compounds have been studied in detail, but much is still lacking in our understanding of the physiological, biochemical, and genetic regulation of their production. Here, we report on the isolation, functional expression, and developmental regulation of Cstps1, a sesquiterpene synthase-encoding gene, involved in citrus aroma formation. The recombinant enzyme encoded by Cstps1 was shown to convert farnesyl diphosphate to a single sesquiterpene product identified as valencene by gas chromatography-mass spectrometry (GC-MS). Phylogenetic analysis of plant terpene synthase genes localized Cstps1 to the group of angiosperm sesquiterpene synthases. Within this group, Cstps1 belongs to a subgroup of citrus sesquiterpene synthases. Cstps1 was found to be developmentally regulated: transcript was found to accumulate only towards fruit maturation, corresponding well with the timing of valencene accumulation in fruit. Although citrus fruits are non-climacteric, valencene accumulation and Cstps1 expression were found to be responsive to ethylene, providing further evidence for the role of ethylene in the final stages of citrus fruit ripening. Isolation of the gene encoding valencene synthase provides a tool for an in-depth study of the regulation of aroma compound biosynthesis in citrus and for metabolic engineering for fruit flavor characteristics.