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Applied and Environmental Microbiology, May 2009, p. 2765-2774, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02681-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Esben H. Hansen,1,¶ Birger Lindberg Møller,2 Gertrud R. Kock,1,¶ Camilla M. Bünner,1,# Charlotte Kristensen,1,¶ Ole R. Jensen,1,{ddagger} Finn T. Okkels,1,§ Carl E. Olsen,3 Mohammed S. Motawia,2 and Jørgen Hansen1*

Poalis A/S, Bülowsvej 25, DK-1870 Frederiksberg C, Denmark,1 Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen,2 Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen3

Received 24 November 2008/ Accepted 6 March 2009

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.

* Corresponding author. Present address: Evolva Biotech A/S, Bülowsvej 25, DK-1870 Frederiksberg C, Denmark. Phone: 45 35 200 240. Fax: 45 35 200 231. E-mail: jorgenh{at}evolva.com

{triangledown} Published ahead of print on 13 March 2009.

Present address: Evolva Biotech A/S, Bülowsvej 25, DK-1870 Frederiksberg C, Denmark.

# Present address: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.

{ddagger} Present address: CPKelco Aps, Ved Banen 16, DK-4623 Lille Skensved, Denmark.

§ Present address: Kongemarken 11, DK-4000 Roskilde, Denmark.

Applied and Environmental Microbiology, May 2009, p. 2765-2774, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02681-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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