Improved vanillin production in baker's yeast through in silico design

<p>Abstract</p> <p>Background</p> <p>Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid <it>Vanilla planifolia</it>. Currently vanillin is mostly produced <it>via </it>chemical synthesis. A <it>de novo </it>synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, <it>Saccharamyces cerevisiae</it>. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.</p> <p>Results</p> <p>Expression of a glycosyltransferase from <it>Arabidopsis thaliana </it>in the vanillin producing <it>S. cerevisiae </it>strain served to decrease product toxicity. An <it>in silico </it>metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (<it>PDC1 </it>and <it>GDH1</it>) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the <it>Δpdc1 </it>mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on <it>de novo </it>vanillin biosynthesis in baker's yeast.</p> <p>Conclusion</p> <p>Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. <it>In vivo </it>verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for <it>in silico </it>design and target selection for improving microbial cell factories.</p

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

Cassava genome from a wild ancestor to cultivated varieties

Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology

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

Crop wild relatives as a genetic resource for generating low-cyanide, drought-tolerant Sorghum

Using a Sorghum bicolor cultivar and seven wild Sorghum species endemic to Australia as our experimental system, we monitored their different responses to drought by assessing growth and morphological, physiological and biochemical parameters. Drought stress significantly decreased height, biomass, the maximum potential quantum efficiency of photosystem II, photosynthetic rate and relative water content in S. bicolor, while several of the wild species were much more tolerant. Drought significantly increased dhurrin concentration in aboveground tissue in S. bicolor but not in the wild species. Root dhurrin content was unaffected by drought in S. bicolor, in contrast to the varied responses observed in the wild species. Sorghum macrospermum and S. brachypodum maintained relatively high growth and photosynthetic performance under drought, with negligible aboveground dhurrin content. These wild species are promising candidates for sorghum crop improvement