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3.
Figure 8

Figure 8. Production of β-amyrin is shown over 48 h for the constructs with single, double and triple over-expressions of Erg8, Erg9, and HFA1, and the control strain.. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

The β-amyrin was detected after the exhaustion of glucose and the initiation of the consumption of ethanol that had produced by the strains. The data shown as total production are means for two independent cultivations for each strain.

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.
4.
Figure 7

Figure 7. Physiological characterization of the reference and recombinant S. cerevisiae strains.. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

(A) Bars represent the growth rates of the constructs relative to the reference strain when grown on glucose and the inset shows the growth curves. (B) Improved in vivo production of ergosterol from CEN.PK constructs. The production yields have been calculated at the end of the exponential growth. During this time period no β-amyrin was detected. Strains: PSY (1023.βΑ), Erg8,PSY (1026.βΑ), Erg9,PSY (1027.βΑ), HFA1,PSY (1029.βΑ), Erg8,Erg9,PSY (1028.βΑ), Erg9,HFA1,PSY (1030.βΑ), Erg8,HFA1,PSY (1057.βΑ), Erg8,Erg9,HFA1,PSY (1031.βΑ).

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.
5.
Figure 5

Figure 5. Ligand binding sites predicted using Q-SiteFinder.. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

The two top ranked binding pockets were selected in each case. The differences between the phosphomevalonate kinase from S. cerevisiae S288C and CEN.PK113-7D strains can be clearly observed from the homology model structures shown. (a) Q-SiteFinder predictions for binding pockets in the phosphomevalonate kinase from Streptococcus pneumoniae, for which crystal structure data is available (PDB ID: 3GON). Q-SiteFinder was able to accurately predict the active site of 3GON with two binding pockets, one each for phosphomevalonate and AMPPNP. Both ligands are represented in stick model. (b) Q-SiteFinder predictions for binding pockets in phosphomevalonate kinase from S. cerevisiae S288C. (c) Q-SiteFinder predictions for binding pockets in phosphomevalonate kinase from S. cerevisiae CEN.PK113-7D.

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.
6.
Figure 6

Figure 6. Systematic gene over-expression in CEN.PK strains harbouring a plasmid (pYES: GAL1 promoter/URA3 selection marker) with the PSY gene (P. sativum) coding for a β-amyrin synthase.. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

The three genes Erg8, Erg9, and HFA1 were ligated in different plasmids with the HIS3, TRP1 and LEU2 selection markers respectively, using the TDH3p promoter, and they were transformed in all combinations (single, double, triple over-expressions) to the respective parental strains leading to prototrophic strains. A visual representation of the final constructs containing from one (1) up to four (4) plasmids, as well as the name of the resulting strains, which is used in the text, is also given.

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.
7.
Figure 4

Figure 4. Ligand interaction diagrams for the active site of the phosphomevalonate kinase (PDB ID: 3GON).. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

It was calculated that only 50% of the charge moieties of the ligands were in van der Waals contact with the protein. (a) The active site residues of phosphomevalonate kinase and their interaction with the ligands phosphomevalonate and AMPPNP through clusters of ordered water. Ligands are shown in ball and stick model. Phosphomevalonate is shown in brown color and AMPPNP in magenta color. Hydrogen bonds are shown as blue dashed lines. (b) Ligand binding pattern for AMPPNP and distance between the interacting amino acid residues calculated using the Accelry Discovery Studio version 2.5. (c) Ligand binding pattern for phosphomevalonate calculated and distance between the interacting amino acid residues using the Accelrys Discovery Studio version 2.5. The values shown are in Å units.

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.
8.
Figure 1

Figure 1. Schematic illustration of the mevalonate, the sterol pathway and the initial step of the fatty acid biosynthetic process, as well as the steps engineered in the current study for triterpene production in yeast.. From: Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers.

The mevalonate pathway is localized to the cytoplasm of eukaryotic cells and supports the biosynthesis of numerous terpenoids using different precursor molecules, while ergosterol is the dominant terpenoid. Whole genome Illumina-Solexa sequencing of CEN.PK113-7D and S288C was completed prior to our study, and SNPs strictly related to metabolic genes were identified [12]. There were clear correlations between physiology and pathway enrichment of non-silent SNPs observed in genes involved in the ergosterol biosynthesis (red font indicates non-silent SNPs, while blue font indicates silent ones), suggesting that genome-sequencing may assist in reducing the genetic target space for metabolic engineering applications. Various combinations of over-expressions (single, double, triple) of genes coding for phosphomevalonate kinase (Erg8), squalene synthase (Erg9), and acetyl-coenzyme A carboxylase (HFA1) may yield yeast strains capable of accumulating excess levels of β-amyrin, a triterpene molecule originating from oxidosqualene.

Karina M. Madsen, et al. PLoS One. 2011;6(3):e14763.

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