<i>In vitro</i> 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

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-, β-, 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.</p

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. <i>O</i>-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

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

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 β-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

Fostering the quality of teaching and learning by developing the "neglected half" of university teachersʼ competencies

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.</jats:p