Jorgen Hansen

De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae)

ABSTRACT Vanillin is one of the worlds 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 worlds 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 bakers 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.

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

BackgroundVanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in bakers yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.ResultsExpression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico 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 (PDC1 and GDH1) 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 Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in bakers yeast.ConclusionUse 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. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

Metabolomic, Transcriptional, Hormonal, and Signaling Cross-Talk in Superroot2

Auxin homeostasis is pivotal for normal plant growth and development. The superroot2 (sur2) mutant was initially isolated in a forward genetic screen for auxin overproducers, and SUR2 was suggested to control auxin conjugation and thereby regulate auxin homeostasis. However, the phenotype was not uniform and could not be described as a pure high auxin phenotype, indicating that knockout of CYP83B1 has multiple effects. Subsequently, SUR2 was identified as CYP83B1, a cytochrome P450 positioned at the metabolic branch point between auxin and indole glucosinolate metabolism. To investigate concomitant global alterations triggered by knockout of CYP83B1 and the countermeasures chosen by the mutant to cope with hormonal and metabolic imbalances, 10-day-old mutant seedlings were characterized with respect to their transcriptome and metabolome profiles. Here, we report a global analysis of the sur2 mutant by the use of a combined transcriptomic and metabolomic approach revealing pronounced effects on several metabolic grids including the intersection between secondary metabolism, cell wall turnover, hormone metabolism, and stress responses. Metabolic and transcriptional cross-talks in sur2 were found to be regulated by complex interactions between both positively and negatively acting transcription factors. The complex phenotype of sur2 may thus not only be assigned to elevated levels of auxin, but also to ethylene and abscisic acid responses as well as drought responses in the absence of a water deficiency. The delicate balance between these signals explains why minute changes in growth conditions may result in the non-uniform phenotype. The large phenotypic variation observed between and within the different surveys may be reconciled by the complex and intricate hormonal balances in sur2 seedlings decoded in this study.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L. DOI: http://dx.doi.org/10.7554/eLife.23001.001