Improved vanillin production in baker's yeast through in silico design

Abstract

Background: Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.

Results: Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast.

Conclusion: Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

Figure 1

Schematic representation of the de novo VG biosynthetic pathway in S. Cerevisisae (as designed by Hansen et al [5]). Metabolites are shown in black, enzymes are shown in black and in italic, cofactors and additional precursors are shown in red. Reactions catalyzed by heterologously introduced enzymes are shown in red. Reactions converting glucose to aromatic amino acids are represented by dashed black arrows. Metabolite secretion is represented by solid black arrows where relative thickness corresponds to relative extracellular accumulation. 3-DSH stands for 3-dedhydroshikimate, PAC stands for protocathechuic acid, PAL stands for protocatechuic aldehyde, SAM stands for S-adenosylmethionine. 3DSD stands for 3-dedhydroshikimate dehydratase, ACAR stands for aryl carboxylic acid reductase, PPTase stands for phosphopantetheine transferase, hsOMT stands for O-methyltransferase, and UGT stands for UDP-glycosyltransferase. Adapted from Hansen et al. [5].

Figure 2

Comparison of targets predicted by OptGene for improved VG productivity. A - Biomass versus VG yield is represented for each knockout mutant phenotype obtained after OptGene simulation using three different reference flux distributions for MOMA. Experimental yields observed for VG0 are represented by the red empty triangle and bar. B/C/D - The predicted VG yield (mol/mol_glc) obtained for each knockout mutant after OptGene simulation using Reference 1/2/3 is given by the length of the coloured bars. For each reference flux distribution, the R³ score was estimated for each of the mutants was calculated and normalized to the mutant presenting the highest value: MA_mut/MA_max*100. 100% represents the mutant with highest R³ score for a given flux distribution. Candidate 80%PDC is not a knockout in silico mutant, rather its PDC reaction is constrained to 80% of the upper bound.

Figure 3

Vanillin β-D-glucoside yield observed for the reference strain (VG0) and metabolically engineered mutants (VG1-4) in batch cultivations. Substrate overall yield for vanillin β-D-glucoside (Y_{S VG}, mg_VG/g_glc), protocatechuic acid (Y_{S PAC}, mg_PAC/g_glc) and protocatechuic aldehyde (Y_{S PAL}, mg_PAL/g_glc) obtained for the reference and engineered strains in batch culture. Pie charts are presented to illustrate relative distribution of PAC, PAL and VG for each strain.

Figure 4

Vanillin β-D-glucoside yield observed for the reference strain (VG0) and metabolically engineered mutants (VG1-4) in continuous cultivations. A - Biomass specific production rate (mg_metab.g_dw^-1.h^-1) for protocatechuic acid (PAC), protocatechuic aldehyde (PAL) and VG in glucose limited chemostat cultivation at dilution rate of 0.1 h-¹. B - Substrate specific yield (Y_{S Metab}, mg_metab/g_glc) for PAC, PAL and VG for strains VG0 and VG2 in glucose limited chemostat cultivation at different dilution rates - 0.1 h-¹(Top) and 0.015 h-¹(*, Bottom).

Figure 5

Flux variability analysis. Reactions were classified based on the comparison of their flux variability range between the reference VG0 and the mutants VG2 and VG4. A- The scheme on the left-hand side illustrates the flux variability ranges defining the six different categories (Blocked and a to e). Flux variability range for the reference strain (VG0) is represented in gray, and the mutant in black. The distribution of VG2 and VG4 reactios among different categories are presented in the bar chart, yellow and red, respectively. B- The reactions from mutant VG2 belonging to categories b and c are further classified accordingly to their metabolic function.

Figure 6

Minimum turnover of selected metabolites from the central carbon metabolism and from the VG biosynthetic pathway (including cofactors). Metabolites from the central carbon metabolism: glucose-6-phosphate, erythrose-4-phosphate, pyruvate and ethanol; Metabolites from the VG biosynthetic pathway (ATP, NADPH, SAM and UDP-glucose). Metabolites for which minimum turnover was calculated are represented by filled circles, metabolites for which no minimum turnover was calculated are represented by open rings. Reactions are represented as arrows. Qualitative variation of the minimum turnovers relatively to the reference (VG0) is shown by the arrows next to each metabolite; yellow corresponds to VG2 while red corresponds to VG4.