Fig 1.
Metabolic engineering approaches in yeast for increased terpenoid production.
A simplified metabolic network of yeast depicting most important reactions is shown. Production of the sesquiterpenoid patchoulol is enabled via expression of patchoulol synthase (via plasmid #1 pSP-P). Overexpression of truncated HMG-CoA reductase (tHMG1, via plasmid #2 pSP-Pt) and fusion of FPP synthase with patchoulol synthase (via plasmid #3 pSP-FPt) to increase the flux from the central carbon precursor acetyl-CoA to the terpenoid product are highlighted in green. The disruption of α-ketoglutarate dehydrogenase (KGD1) to redirect the metabolic flux from citric acid cycle via pyruvate dehydrogenase bypass towards terpenoids is shown in red. Overexpression of ATP-citrate lyase (via plasmid #4 pSP-FPt-ACL) to produce cytosolic acetyl-CoA via pyruvate dehydrogenase and citric acid cycle instead of pyruvate dehydrogenase bypass is shown in blue.
Table 1.
Plasmids used in this study.
Fig 2.
Sesquiterpenoid spectrum of patchoulol synthase produced by yeast.
A representative GC/FID analysis of the patchoulol synthase products in the organic phase of a yeast two-phase cultivation heterologously expressing patchoulol synthase is shown. 22 Peaks with nominal masses of 222 (sesquiterpenoids with hydroxy-group) and 204 (sesquiterpenoids without hydroxy-group) were detected. The peak marked with an asterisk (*) was identified as patchoulol.
Fig 3.
Effect of tHMG1 overexpression and fusion of FPP synthase and patchoulol synthase on terpenoid production.
Terpenoid formation of yeast strains carrying pSP-P, pSP-Pt and pSP-FPt after growth on glucose in batch conditions: (a) ergosterol content, (b) sesquiterpenoid yield on glucose and (c) squalene content. Shown are mean values and standard deviations of six experiments.
Fig 4.
Effect of carbon source on terpenoid production.
Terpenoid production of yeast strain carrying pSP-FPt during glucose-phase (cells were harvested as soon as glucose was exhausted) and during ethanol-phase (cells were harvested after four days when ethanol, glycerol and acetate were exhausted). (a) sesquiterpenoid titer; (b) ergosterol, squalene and sesquiterpenoid content per cell dry weight, CDW; (c) fraction of sesquiterpenoids in total terpenoids (ergosterol, squalene and sesquiterpenoids). Shown are mean values and standard deviations of three experiments.
Fig 5.
Effect of disruption of KGD1 on physiological parameters of yeast.
Influence of KGD1 disruption (▲, kgd1Δ) in comparison to the wild type (○) on: (a) biomass formation (cell dry weight, CDW), (b) ethanol formation, (c) glycerol formation and (d) acetate formation as a function of time during glucose-phase as well as ethanol-phase, arrows indicate the point in time when glucose was exhausted. Shown are mean values and standard deviations of three experiments.
Fig 6.
Effect of disruption of KGD1 on terpenoid production.
Influence of KGD1 disruption in comparison to the wild type on: (a) ergosterol content, (b) squalene content, (c) sesquiterpenoid titer and (d) sesquiterpenoid yield on biomass during glucose-phase (glc-phase) and ethanol-phase at four and six days (4 d, 6 d). Shown are mean values and standard deviations of three experiments.
Fig 7.
Western blot analysis of yeast proteins.
A representative western blot of protein from yeast strain carrying pSP-FPt-ACL (ACL) and pSP-FPt as control (C) is shown. The protein ladder is shown on the left (M). The predicted position of AclA-1-T2A is indicated with an arrow.
Fig 8.
Effect of ATP-citrate lyase (ACL) overexpression on terpenoid production.
Terpenoid formation, i.e. (a) ergosterol content, (b) squalene content, (c) sesquiterpenoid titer and (d) sesquiterpenoid yield on biomass of yeast strains carrying pSP-FPt and pSP-FPt-ACL during glucose-phase (glc-phase) and ethanol-phase at four and six days (4 d, 6 d). Shown are mean values and standard deviations of three experiments.