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 UDPglycosyltransferase, converting vanillin into vanillin ␤-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.

A novel principle for selection of transgenic plant cells is presented. In contrast to traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), this selection method actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. Therefore, this selection strategy is termed 'positive selection'. TheE. coli β-glucuronidase gene was used as selectable (as well as screenable) gene and a glucuronide derivative of the cytokinin benzyladenine as selective agent which is inactive as cytokinin but, upon hydrolysis by GUS, active cytokinin is released stimulating the transformed cells to regenerate. Selection ofAgrobacterium tumefaciens inoculated of tobacco leaf discs on benzyladenine N-3-glucuronide (7.5-15 mg/l) resulted in 1.7-2.9 fold higher transformation frequencies compared to kanamycin selection. A significant advantage of this selection procedure is the elimination of the need for herbicide and antibiotic resistance genes.

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BackgroundBlack carrots (Daucus carota ssp. sativus var. atrorubens Alef.) constitute a valuable source of anthocyanins, which are used as natural red, blue and purple food colourants. Anthocyanins and phenolic compounds are specialised metabolites, accumulation of which often requires elicitors, which act as molecular signals in plant stress responses. In the present study, ethephon, an ethylene-generating compound was explored as enhancer of anthocyanin and phenolic contents during growth of ‘Deep Purple’ black carrots. The effects of ethephon on several parameters were investigated, and the expression of biosynthetic anthocyanin genes was studied during growth and anthocyanin accumulation.ResultsRoots of ethephon-treated carrot plants exhibited an increase in anthocyanin content of approximately 25%, with values ranging from 2.25 to 3.10 mg g−1 fresh weight, compared with values ranging from 1.50 to 1.90 mg g−1 fresh weight in untreated roots. The most rapid accumulation rate for anthocyanins, phenolic compounds, soluble solids and dry matter was observed between 10 and 13 weeks after sowing in both untreated and ethephon-treated carrots. The differences in anthocyanin contents between untreated and treated carrots increased for several weeks after the ethephon treatment was terminated. Five cyanidin-based anthocyanin forms were identified, with variable relative abundance values detected during root growth. Overall, the expression of the anthocyanin biosynthetic genes analysed (PAL1, PAL3, F3H1, DFR1, LDOX2) increased in response to ethephon treatment, as did the expression of the MYB1 transcription factor, which is associated with activation of the phenylpropanoid pathway under stress conditions. In addition, a correlation was proposed between ethylene and sugar contents and the induction of anthocyanin synthesis.ConclusionsThis study presents a novel method for enhancing anthocyanin content in black carrots. This finding is of economic importance as increased pigment concentration per unit of biomass implies improved profitability parameters in food colour production. We provide new insight into the accumulation patterns of the different cyanidin-based anthocyanins and phenolic compounds during root growth. Moreover, we show that enhanced anthocyanin content in ethephon-treated carrots is accompanied by increased expression of anthocyanin biosynthetic genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1021-7) contains supplementary material, which is available to authorized users.

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