In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo. Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production. Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to meet the required activities for an increased product formation in vivo.