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

Abstract

ABSTRACT 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.