In this study, we demonstrate complete de novo vanillin production outside the Vanilla planifolia seed pod or other plants. This represents the first example of one-cell microbial generation of this valuable compound from glucose, at a production level scalable to industrial needs. The capability for vanillin biosynthesis was introduced into two common yeast species, Schizosaccharomyces pombe and Saccharomyces cerevisiae. The heterologous pathway for vanillin biosynthesis was engineered in both organisms by the expression of three genes, one from a mold, one from a bacterium, and one of human origin, and in the case of S. cerevisiae, one additional bacterial gene. We obtained a vanillin production of 65 and 45 mg/liter in S. pombe and S. cerevisiae, respectively, free of contaminating isomers, without any specific optimization of media and growth conditions. Although vanillin biosynthesis was less efficient in S. cerevisiae than in S. pombe, our data actually indicate a higher vanillin production potential in S. cerevisiae, since the combined production of vanillin and its precursors and metabolites was almost twice as high with S. cerevisiae as with S. pombe (Table ). The accumulated levels of the various metabolites indicate that more dehydroshikimic acid is converted to protecatechuic acid in our S. cerevisiae experiment but also that about the same proportion of this (70% for S. cerevisiae, 75% for S. pombe) is reduced by the introduced ACAR enzyme. The reason for the lower production of vanillin in S. cerevisiae is a higher ability of this organism to reduce vanillin to its corresponding alcohol. This undesired property of S. cerevisiae became obvious at the beginning of the project and was addressed by inactivation of the ADH6-encoded alcohol dehydrogenase. In the set of experiments undertaken to identify the importance of different alcohol dehydrogenases in vanillin reduction, a modest effect of inactivation of several other genes (e.g., ADH7) was registered, and it is likely that inactivation of additional alcohol dehydrogenases in the S. cerevisiae vanillin producer would result in a significant increase in vanillin production.
The observation that nearly identical proportions of the biosynthesized protocatechuic acid were reduced by both yeast strains demonstrates that introduction of the C. glutamicum
PPTase gene in our S. cerevisiae
vanillin producer resulted in an activation of the ACAR enzyme to the same level as that seen in S. pombe
. It is indeed puzzling that bacterial ACAR can be activated by inherent enzymes in one yeast but not in another. Enzymes requiring phosphopantetheinylation for activation are not abundant in these yeast species, but one well-known example present in both is α-aminoadipate reductase. Both species carry a known PPTase activity taking care of this (Lys5p in S. cerevisiae
, Lys7p in S. pombe
), and these are obvious candidates for heterologous ACAR activation (though another could be the PPTase activating mitochondrial fatty acid synthase). A plausible explanation for the differences in PPTase activity in the two yeasts is derived from the following observations (10
). Whereas S. pombe
α-aminoadipate synthase can be activated by PPTases present in E. coli
, this is not the case for α-aminoadipate synthase from Candida albicans
. The C. albicans
enzyme is much more closely related to the S. cerevisiae
enzyme than to the S. pombe
enzyme. Turning the argument around, this may imply that S. pombe
(via its lys7+
-encoded PPTase), but not S. cerevisiae
, has the inherent ability to activate the bacterial ACAR enzyme. Not surprisingly, a PPTase from Corynebacterium glutamicum
, a high-GC, gram-positive bacterium related to Nocardia
sp., turned out to be the most efficient in ACAR activation.
As previously outlined, vanillin β-d-glucoside is the storage form of vanillin found in the Vanilla pod. It is nontoxic to most organisms, including yeast, and has a higher solubility in water than does vanillin. In addition, the formation of vanillin β-d-glucoside most likely pulls the biosynthesis further in the direction of vanillin production. The Arabidopsis thaliana UDP-glucose glycosyltransferase UGT72E2 exhibited high substrate specificity toward vanillin. In concordance with this observation, its expression in the vanillin-producing S. pombe strain resulted in almost all vanillin being converted into vanillin β-d-glucoside. The ability to turn vanillin into vanillin β-d-glucoside in vivo is very important, because microbial production of nonglucosylated vanillin beyond the 0.5- to 1-g/liter scale would be hampered by the toxicity of free vanillin. Glucosylation would serve to circumvent the inhibitory effect. Although glucosylation did not give rise to a major increase in vanillin production, the content of nonmethylated intermediates (protocatechuic acid and aldehyde) was reduced by more than 50% (Fig. ). This indicates that glucosylation does indeed drive production of methylated vanillin equivalents, but that only a certain amount of dehydroshikimic acid is available during the period of time when our introduced vanillin pathway is active. There could be many reasons for this and we are currently studying several possibilities.
“Sustainable” and “renewable” biological production systems are attracting a lot of attention these days, due to the global warming issue and associated interest in developing a chemical industry that is independent of fossil fuel starting materials; thus, “white biotechnology” is having a tremendous comeback. S. cerevisiae
is a very attractive production organism in white biotechnology, because this yeast species is well characterized, is easy to manipulate and grow, and has gained GRAS status. Metabolic engineering of S. cerevisiae
has resulted in very high yields of certain primary yeast metabolites, e.g., 153 g/liter of pyruvate (43
), but de novo productivities of novel metabolites have usually been quite modest, ranging from 153 mg/liter (the terpenoid amorphadiene [38
]) to only just detectable amounts (e.g., the polyketide precursor methylmalonyl-coenzyme A [26
]) (reviewed in reference 28
). To our knowledge, our study is the first in which aromatic amino acid biosynthesis intermediates are used for production of a novel compound, and in that perspective, we find our initial productivity of 45 mg/liter satisfactory. We are aware, however, that even though the market prices for “natural” vanillin and for vanillin-β-d
-glucoside are high, the biological production system presented here needs to be improved significantly to offer a truly sustainable alternative. It was recently shown that simple genetic modifications may increase the metabolic flux through the S. cerevisiae
aromatic amino acid biosynthesis pathway 4.5-fold and the extracellular concentration of shikimic acid (the direct metabolite of dehydroshikimic acid) more than 200-fold (22
). This provides obvious opportunities for significant future increases in vanillin production using yeasts as production organisms.