Overexpression of ispG to alleviate efflux of MEC for lycopene production.

<p>SIDF: BL21 Gold (DE3) harboring pET-SIDF and pACLYC; SIDFG: BL21 Gold (DE3) harboring pET-SIDFG and pACLYC. (A) Lycopene production as a function of time; (B) Extracellular MEC concentration as a function of time; (C) Intracellular MEC concentration as a function of time; (D) Intracellular HMBPP concentration as a function of time. Presented data were average of triplicates and standard errors were drawn on the plot.</p

Effects of UPLC gradient on chromatography separation of the DXP pathway intermediates.

<p>Different concentrations (0.8, 0.4, 2.6, 2.6, 0.5 and 2 µM respectively) of DXP, MEP, CDP-ME, CDP-MEP, MEC, and HMBPP were prepared in 1 mL acidic extraction solution and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#s4" target="_blank">METHODS AND MATERIALS</a>. Quantification m/z ratio for each compound was extracted from total ion chromatography as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t001" target="_blank">Table 1</a> and traces were overlaid. (A) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 40% aqueous solution was used in step 3 and 4; (B) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 50% aqueous solution was used in step 3 and 4; (C) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a>; (D) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 70% aqueous solution was used in step 3 and 4; (E) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 80% aqueous solution was used in step 3 and 4.</p

The performance of different combinations of DXP pathway genes in <i>E</i>. <i>coli</i>.

<p>(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown. </p

The effects of Fe-S operons on the amorphadiene production.

<p>Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors of four replicates were presented as error bars. The two tailed p-values of student’s t-test were carried out to compare certain conditions and presented as P in the figure.</p

The cross-lapping in vitro assembly (CLIVA) method.

<p>(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid. </p

Analysis of bacteria other than <i>E. coli</i>.

<p>Intracellular DXP pathway intermediates at middle exponential growth phase. Presented data were average of triplicates and standard errors were drawn on the plot. <i>C. violaceum</i>: <i>Chromobacterium violaceum; P. aeruginosa : Pseudomonas aeruginosa; B. subtilis : Bacillus subtilis</i>.</p

The effects of fosmidomycin inhibition of dxr on DXP metabolism.

<p>Twenty five microgram per milliliter fosmidomycin was used to inhibit MEC biosynthesis at 4 h after induction, and the metabolites of BL21 Gold (DE3) harboring pET-SIDF and pAC-LYC were analyzed before and 2 h after inhibition; Presented data were average of triplicates and standard errors were drawn on the plot.</p

Mobile phase gradient used for the separation of DXP intermediates in the UPLC method.

*<p>Aqueous solution: 15 mM acetic acid and 10 mM tributylamine.</p

Analytical performance of the SPE UPLC-MS method.

*<p>Concentration of CDP-MEP for intra-day/inter-day variation was 0.25 µM.</p

Efflux pumps were not repressed in the strain overexpressing dxs-idi-ispDF-ispG.

<p>Transcription fold change of efflux pump encoding genes upon ispG overexpression; bcr, mdtJ, mdtH, mdtA, mdtG, cmr, emrK, emrE, fsr, mdtL and acrA were known genes encoding <i>E. coli</i> efflux pumps. Transcriptions of the following efflux pump encoding genes were too low to be accurately quantified in this study: cusC, macA, mdtK, mdtA, mdtM and emrD. They were reported not to be expressed in regular aerobic conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone.0047513-Zhang2" target="_blank">[24]</a>. Presented data were average of triplicates and standard errors were drawn on the plot.</p