Improving fatty acid availability for bio-hydrocarbon production in Escherichia coli by metabolic engineering.

Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass (in the order of cyoA, nuoA, ndh, adhE, dld, pta, and iclR) to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation

GC chromatograms of triacylglycerol from strains BL 21 Star^TM DE3, △<i>dgkA</i>, SCO0958, △<i>dgkA</i>/SCO0958, △<i>dgkA</i>/ATF1, △<i>dgkA</i>/ATF2, △<i>dgkA</i>/WS1 and △<i>dgkA</i>/WS2.

<p>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.</p

Characterization of <i>E. coli</i> strains with genetic modifications in central carbon metabolism.

<p>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.</p

Total amounts of fatty acids and triacylglycerol (A) in strains BL 21 Star^TM DE3, △<i>dgkA</i>, SCO0958, △<i>dgkA</i>/SCO0958, △<i>dgkA</i>/WS1, and △<i>dgkA</i>/WS2 were measured individually by GC-FID.

<p>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.</p

Overview of genetically modified metabolic pathways designed for increasing fatty acid production.

<p>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 <i>iclR</i>.</p

The total fatty acid content in <i>E. coli</i> strains with genetic modifications in both central carbon metabolism and fatty acid biosynthesis pathway (A) and the corresponding fatty acid composition (B).

<p>The strains were cultured in M9 minimal medium with 2% glucose for 48 hrs. BL 21: BL 21 Star^TM DE3; 5△: △<i>cyoA</i>△<i>adhE</i>△<i>nuoA</i>△<i>ndh</i>△<i>pta</i>; 6△: △<i>cyoA</i>△<i>adhE</i>△<i>nuoA</i>△<i>ndh</i>△<i>pta</i>△<i>dld</i>; 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.</p

Total amounts of fatty acids and triacylglycerol in strain △<i>dgkA</i>/WS1 cultured in the minimal medium M9, rich medium LB, and LB supplemented with different concentrations of glucose and sodium bicarbonate.

<p>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.</p

Direct Observation of Confinement Effects of Semiconducting Polymers in Polymer Blend Electronic Systems

Abstract The advent of special types of polymeric semiconductors, known as “polymer blends,” presents new opportunities for the development of next‐generation electronics based on these semiconductors' versatile functionalities in device applications. Although these polymer blends contain semiconducting polymers (SPs) mixed with a considerably high content of insulating polymers, few of these blends unexpectedly yield much higher charge carrier mobilities than those of pure SPs. However, the origin of such an enhancement has remained unclear owing to a lack of cases exhibiting definite improvements in charge carrier mobility, and the limited knowledge concerning the underlying mechanism thereof. In this study, the morphological changes and internal nanostructures of polymer blends based on various SP types with different intermolecular interactions in an insulating polystyrene matrix are investigated. Through this investigation, the physical confinement of donor–acceptor type SP chains in a continuous nanoscale network structure surrounded by polystyrenes is shown to induce structural ordering with more straight edge‐on stacked SP chains. Hereby, high‐performance and transparent organic field‐effect transistors with a hole mobility of ≈5.4 cm2 V–1 s–1 and an average transmittance exceeding 72% in the visible range are achieved