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

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 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.

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…Only a small number of truly de novo pathway designs have been published and most use isolated heterologous enzymes acting on their cognate substrates [18][19][20]50 . Engineered pathways to liquid fuels, in particular, have predominantly relied on entirely natural (ethanol, butanol, isoprenoid) or terminally modified natural pathways (FAS, amino-acid aKAE, isoprenoid).…”

“…In some cases, pathways have been repurposed to synthesize new products by capitalizing on the natural capacity of enzymes to accept closely related substrates or by engineering protein specificity [15][16][17] . The boldest designs have utilized previously undescribed pathways created by combining the natural chemistry of individual enzymes from multiple hosts [18][19][20] .…”

Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network

“…For example, a heterologous pathway that produces amorphadiene, a precursor of the antimalarial drug artemisinin, in E. coli results in accumulation of 3-hydroxy-3-methyl-glutarylCoA and severe growth inhibition (20). Indole, an intermediate in an engineered pathway for synthesis of taxadiene (9), and vanillin, formed via a heterologous pathway in yeast, both cause substantial inhibition of growth (5). The targets of toxicity were not identified in any of these cases, although considerable effort was invested in the case of the amorphadiene synthesis pathway.…”

“…Often, "green" processes rely on introduction of novel metabolic pathways patched together from enzymes that have some ability to catalyze the needed transformations, even if this is not their primary activity, into suitable microbial hosts. Synthetic biologists have generated metabolic pathways that do not exist in nature for synthesis of platform chemicals such as isobutyric acid (4), specialty chemicals such as vanillin (5), and a variety of secondary metabolites that have potential uses as therapeutic agents (6,7). Notable successes include the microbial syntheses of amorphadiene (8), a precursor of the antimalarial drug artemisinin, and taxadiene, the precursor of the potent anticancer drug Taxol (9).…”

Effects of Active‐Site Modification and Quaternary Structure on the Regioselectivity of Catechol‐O‐Methyltransferase

“…Nature has evolved regiocomplementary enzymes for certain transformations;2 however, where no suitable native enzyme exists, engineering regiocomplementary variants to deliver defined regioisomeric products is an enticing prospect. One example where the lack of enzyme regioselectivity limits potential commercial productivity is the engineered biosynthesis of the flavor and fragrance agent vanillin 2 a 3. Catechol‐ O ‐methyltransferase (COMT) has been used to methylate 3,4‐dihydroxybenzaldehyde (DHBAL, 1 a ) and 3,4‐dihydroxybenzoic acid (DHBA, 1 b ) in engineered strains of Escherichia coli and fission yeast for the production of vanillin 2 a from glucose (Figure 1 A and Figure S1 in the Supporting Information) 3, 4.…”