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.
Hansen
Esben H
EH
Poalis A/S, DK-1870 Frederiksberg C, Denmark.
Møller
Birger Lindberg
BL
Kock
Gertrud R
GR
Bünner
Camilla M
CM
Kristensen
Charlotte
C
Jensen
Ole R
OR
Okkels
Finn T
FT
Olsen
Carl E
CE
Motawia
Mohammed S
MS
Hansen
Jørgen
J
D000426:Alcohol Dehydrogenase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001547:Benzaldehydes / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D017353:Gene Deletion; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics
D001426:Bacterial Proteins; D001547:Benzaldehydes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C082662:phosphopantetheinyl transferase; C100058:vanillin; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; C446927:ADH6 protein, S cerevisiae; C019825:carboxylic acid reductase; D008780:Methyltransferases; D016695:Glycosyltransferases; D017855:Transferases (Other Substituted Phosphate Groups); C044921:3-dehydroshikimate dehydratase; D006836:Hydro-Lyases; D005947:Glucose