Disentangling hydroxynitrile glucoside biosynthesis in a barley (Hordeum vulgare) metabolon provides access to elite malting barleys for ethyl carbamate-free whisky production.

Barley produces several specialized metabolites, including five α-, ß-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production.

Biosynthesis and biotechnological production of the anti-obesity agent celastrol.

Obesity is a major health risk still lacking effective pharmacological treatment. A potent anti-obesity agent, celastrol, has been identified in the roots of Tripterygium wilfordii. However, an efficient synthetic method is required to better explore its biological utility. Here we elucidate the 11 missing steps for the celastrol biosynthetic route to enable its de novo biosynthesis in yeast. First, we reveal the cytochrome P450 enzymes that catalyse the four oxidation steps that produce the key intermediate celastrogenic acid. Subsequently, we show that non-enzymatic decarboxylation-triggered activation of celastrogenic acid leads to a cascade of tandem catechol oxidation-driven double-bond extension events that generate the characteristic quinone methide moiety of celastrol. Using this acquired knowledge, we have developed a method for producing celastrol starting from table sugar. This work highlights the effectiveness of combining plant biochemistry with metabolic engineering and chemistry for the scalable synthesis of complex specialized metabolites.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate.

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.

Synthesis of 11-carbon terpenoids in yeast using protein and metabolic engineering.

One application of synthetic biology is the redesign of existing biological systems to acquire new functions. In this context, expanding the chemical code underlying key biosynthetic pathways will lead to the synthesis of compounds with new structures and potentially new biological activities. Terpenoids are a large group of specialized metabolites with numerous applications. Yet, being synthesized from five-carbon units, they are restricted to distinct classes that differ by five carbon atoms (C10, C15, C20, etc.). To expand the diversity of terpenoid structures, we engineered yeast cells to synthesize a noncanonical building block with 11 carbons, and produced 40 C11 terpene scaffolds that can form the basis for an entire terpenoid class. By identifying a single-residue switch that converts C10 plant monoterpene synthases to C11-specific enzymes, we engineered dedicated synthases for C11 terpene production. This approach will enable the systematic expansion of the chemical space accessed by terpenoids.

Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering.

Almond and sweet cherry are two economically important species of the Prunus genus. They both produce the cyanogenic glucosides prunasin and amygdalin. As part of a two-component defense system, prunasin and amygdalin release toxic hydrogen cyanide upon cell disruption. In this study, we investigated the potential role within prunasin and amygdalin and some of its derivatives in endodormancy release of these two Prunus species. The content of prunasin and of endogenous prunasin turnover products in the course of flower development was examined in five almond cultivars - differing from very early to extra-late in flowering time - and in one sweet early cherry cultivar. In all cultivars, prunasin began to accumulate in the flower buds shortly after dormancy release and the levels dropped again just before flowering time. In almond and sweet cherry, the turnover of prunasin coincided with increased levels of prunasin amide whereas prunasin anitrile pentoside and ß-D-glucose-1-benzoate were abundant in almond and cherry flower buds at certain developmental stages. These findings indicate a role for the turnover of cyanogenic glucosides in controlling flower development in Prunus species.

Bottom-Up Elucidation of Glycosidic Bond Stereochemistry.

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/ß glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish α- and ß-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

General and Stereocontrolled Approach to the Chemical Synthesis of Naturally Occurring Cyanogenic Glucosides.

An effective method for the chemical synthesis of cyanogenic glucosides has been developed as demonstrated by the synthesis of dhurrin, taxiphyllin, prunasin, sambunigrin, heterodendrin, and epiheterodendrin. O-Trimethylsilylated cyanohydrins were prepared and subjected directly to glucosylation using a fully acetylated glucopyranosyl fluoride donor with boron trifluoride-diethyl etherate as promoter to afford a chromatographically separable epimeric mixture of the corresponding acetylated cyanogenic glucosides. The isolated epimers were deprotected using a triflic acid/MeOH/ion-exchange resin system without any epimerization of the cyanohydrin function. The method is stereocontrolled and provides an efficient approach to chemical synthesis of other naturally occurring cyanogenic glucosides including those with a more complex aglycone structure.

Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata.

Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a γ-hydroxynitrile glucoside structurally related to cyanogenic glucosides. Sinigrin may defend this plant against a broad range of enemies, while alliarinoside confers resistance to specialized (glucosinolate-adapted) herbivores. Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species. Administration of [UL-(14)C]-methionine to excised leaves of A. petiolata showed that both alliarinoside and sinigrin were biosynthesized from methionine. The biosynthesis of alliarinoside was shown not to bifurcate from sinigrin biosynthesis at the oxime level in contrast to the general scheme for hydroxynitrile glucoside biosynthesis. Instead, the aglucon of alliarinoside was formed from metabolism of sinigrin in experiments with crude extracts, suggesting a possible biosynthetic pathway in intact cells. Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution. Metabolite profiling by LC-MS showed no evidence of the presence of cyanogenic glucosides in A. petiolata. However, we detected hydrogen cyanide (HCN) release from sinigrin and added thiocyanate ion and benzyl thiocyanate in A. petiolata indicating an enzymatic pathway from glucosinolates via allyl thiocyanate and indole glucosinolate derived thiocyanate ion to HCN. Alliarinoside biosynthesis and HCN release from glucosinolate-derived metabolites expand the range of glucosinolate-related defenses and can be viewed as a third line of defense, with glucosinolates and thiocyanate forming protein being the first and second lines, respectively.

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species.

Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and high resolution MS (HR-MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and 'anitriles' derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.

In vitro Biochemical Characterization of All Barley Endosperm Starch Synthases.

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava.

Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l-valine and l-isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside-specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2-hydroxy-2-methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co-occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co-expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine-tuning nitrogen assimilation in cassava.

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway.

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific ß-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.

Tissue and cellular localization of individual beta-glycosidases using a substrate-specific sugar reducing assay.

Traditional methods to localize beta-glycosidase activity in tissue sections have been based on incubation with the general substrate 6-bromo-2-naphthyl-beta-d-glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6-bromo-2-naphthyl-beta-d-glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different beta-glycosidases present in the tissue. To be able to monitor the occurrence of individual beta-glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any beta-glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5-triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two beta-glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.

De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae).

Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin beta-D-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.

Starch phosphorylation--maltosidic restrains upon 3'- and 6'-phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy.

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.

Chemical synthesis of a dual branched malto-decaose: a potential substrate for alpha-amylases.

A convergent block strategy for general use in efficient synthesis of complex alpha-(1-->4)- and alpha-(1-->6)-malto-oligosaccharides is demonstrated with the first chemical synthesis of a malto-oligosaccharide, the decasaccharide 6,6''''-bis(alpha-maltosyl)-maltohexaose, with two branch points. Using this chemically defined branched oligosaccharide as a substrate, the cleavage pattern of seven different alpha-amylases were investigated. Alpha-amylases from human saliva, porcine pancreas, barley alpha-amylase 2 and recombinant barley alpha-amylase 1 all hydrolysed the decasaccharide selectively. This resulted in a branched hexasaccharide and a branched tetrasaccharide. Alpha-amylases from Asperagillus oryzae, Bacillus licheniformis and Bacillus sp. cleaved the decasaccharide at two distinct sites, either producing two branched pentasaccharides, or a branched hexasaccharide and a branched tetrasaccharide. In addition, the enzymes were tested on the single-branched octasaccharide 6-alpha-maltosyl-maltohexaose, which was prepared from 6,6''''-bis(alpha-maltosyl)-maltohexaose by treatment with malt limit dextrinase. A similar cleavage pattern to that found for the corresponding linear malto-oligosaccharide substrate was observed.

Hydration of the amylopectin branch point. Evidence of restricted conformational diversity of the alpha-(1-->6) linkage.

The hydration behavior of a model compound for the amylopectin branch point, methyl 6'-alpha-maltosyl-alpha-maltotrioside, was investigated by combining molecular dynamics simulations in explicit water, 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements, and exploratory multivariate data analysis. In comparison with results on a tetrasaccharide analogue, the study reveals that the conformational diversity of the three-bond alpha-(1-->6) linkage becomes quite limited in aqueous solution upon the addition of a fifth glucose residue that elongates the alpha-(1-->6) branch. This investigation reveals two plausible starch branch point structures, one that permits the formation of double helices and one that is adapted for interconnection of double helices. The apparent rigidity of the former is explained by the presence of water pockets/bridges in the vicinity of the branch point that lock the pentasaccharide structure into one conformational family that is able to accommodate the creation of the double-helical amylopectin structure.

A hydration study of (1-->4) and (1-->6) linked alpha-glucans by comparative 10 ns molecular dynamics simulations and 500-MHz NMR.

The hydration behavior of two model disaccharides, methyl-alpha-D-maltoside (1) and methyl-alpha-D-isomaltoside (2), has been investigated by a comparative 10 ns molecular dynamics study. The detailed hydration of the two disaccharides was described using three force fields especially developed for modeling of carbohydrates in explicit solvent. To validate the theoretical results the two compounds were synthesized and subjected to 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements (1: 4.0. 10(-6) cm(2). s(-1); 2: 4.2. 10(-6) cm(2). s(-1)). In short, the older CHARMM-based force field exhibited a more structured carbohydrate-water interaction leading to better agreement with the diffusional properties of the two compounds, whereas especially the alpha-(1-->6) linkage and the primary hydroxyl groups were inaccurately modeled. In contrast, the new generation of the CHARMM-based force field (CSFF) and the most recent version of the AMBER-based force field (GLYCAM-2000a) exhibited less structured carbohydrate-water interactions with the result that the diffusional properties of the two disaccharides were underestimated, whereas the simulations of the alpha-(1-->6) linkage and the primary hydroxyl groups were significantly improved and in excellent agreement with homo- and heteronuclear coupling constants. The difference between the two classes of force field (more structured and less structured carbohydrate-water interaction) was underlined by calculation of the isotropic hydration as calculated by radial pair distributions. At one extreme, the radial O em leader O pair distribution function yielded a peak density of 2.3 times the bulk density in the first hydration shell when using the older CHARMM force field, whereas the maximum density observed in the GLYCAM force field was calculated to be 1.0, at the other extreme.