Figure 1.
Overview of genetically modified metabolic pathways designed for increasing fatty acid production.
Color codes: the orange block contains the triacylglycerol biosynthesis pathway; the green block contains gene manipulations for removal competing pathways in central carbon metabolism; the turquoise block contains the TCA cycle and the glyoxylate bypass pathway, which is shown in green arrows and activated through knockout of the regulatory repressor gene iclR.
Figure 2.
Characterization of E. coli strains with genetic modifications in central carbon metabolism.
The strains were cultured in M9 minimal medium with 2% glucose for 48 hrs. The total amount of fatty acids (A) and the fatty acid composition (B) were quantified by GC-FID. Other fermentation properties were determined, including the final concentration of by-products lactate and acetate (C), the final cell density (OD), and the final glucose concentration (D). Data presented are averages of two replicate cultures and error bars represent the standard error.
Figure 3.
The total fatty acid content in E. coli strains with genetic modifications in both central carbon metabolism and fatty acid biosynthesis pathway (A) and the corresponding fatty acid composition (B).
The strains were cultured in M9 minimal medium with 2% glucose for 48 hrs. BL 21: BL 21 StarTM DE3; 5△: △cyoA△adhE△nuoA△ndh△pta; 6△: △cyoA△adhE△nuoA△ndh△pta△dld; TE: a leaderless version of TesA that is targeted to the cytosol; ACC: Acetyl-CoA Carboxylase. Data presented are averages of two replicate cultures and error bars represent the standard error.
Figure 4.
GC chromatograms of triacylglycerol from strains BL 21 StarTM DE3, △dgkA, SCO0958, △dgkA/SCO0958, △dgkA/ATF1, △dgkA/ATF2, △dgkA/WS1 and △dgkA/WS2.
All strains were cultured in LB at 30 °C. The location of the TAG peaks is indicated in the figure. Tricarpin is used as an internal standard.
Figure 5.
Total amounts of fatty acids and triacylglycerol (A) in strains BL 21 StarTM DE3, △dgkA, SCO0958, △dgkA/SCO0958, △dgkA/WS1, and △dgkA/WS2 were measured individually by GC-FID.
The fatty acid compositions of these strains were also determined (B). All strains were cultured in LB at 30 °C for 48 hrs. Data presented are averages of two replicate cultures and error bars represent the standard error.
Figure 6.
Total amounts of fatty acids and triacylglycerol in strain △dgkA/WS1 cultured in the minimal medium M9, rich medium LB, and LB supplemented with different concentrations of glucose and sodium bicarbonate.
LB 1-2: LB supplemented with 1% glucose and 2 g/L sodium bicarbonate; LB 2-8: LB supplemented with 2% glucose and 8 g/L sodium bicarbonate; LB 5-10: LB supplemented with 5% glucose and 10 g/L sodium bicarbonate. Data presented are averages of two replicate cultures and error bars represent the standard error.
Figure 7.
Total amounts of fatty acids (A) and triacylglycerol (B) in strains containing both central carbon metabolism modifications and the triacylglycerol biosynthesis pathway.
6△ refers to six knockouts of △cyoA△adhE△nuoA△ndh△pta△dld; 7△ combines 6△ and △iclR. Two culture media, LB 1-2 and LB 5-10, were tested. All strains were cultured at 30 °C for 48 hrs. Data presented are averages of two replicate cultures and error bars represent the standard error.