Substrate consumption, isoprene and biomass productions from different feeding modules^a.

a<p>Module 1, 2 and 3 used 10 g L⁻¹ glucose as substrate; module 4 and 5 used 10 g L⁻¹ D-xylose as substrate. Data reported were average values of duplicate cultivation runs.</p

Four glycolytic pathways present in <i>E. coli</i>.

<p>EMP, Embden-Meyerhof pathway; PPP, pentose phosphate pathway; EDP, Entner-Doudoroff pathway.</p

Pyruvate and G3P generation, energy and reducing equivalents production of different glycolytic pathways.

<p>Pyruvate and G3P generation, energy and reducing equivalents production of different glycolytic pathways.</p

Participation of MEP-dependent isoprene biosynthesis pathway into two modules.

<p>Gene symbols and the enzymes they encode (all genes were from <i>E. coli</i> except where noted): <i>dxs</i>, DXP synthase; <i>ispC</i>, DXP reductionisomerase; <i>ispD</i>, DXP-ME synthase; <i>ispE</i>, CDP-ME kinase; <i>ispF</i>, MECPP synthase; <i>ispG</i>, HMBPP synthase; <i>ispH</i>, HMBPP reductase; <i>idi</i>, IPP isomerase; <i>ispS</i>, isoprene synthase (<i>P. alba</i>). Pathway intermediates: G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-<i>C</i>-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-<i>C</i>-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2-<i>C</i>-methyl-D-erythritol 2-phosphate; MECPP, 2-<i>C</i>-methyl-D-erythritol 2,4-cyclopyrophosphate; HMBPP, 1-hydroxy-2-methyl-2-(<i>E</i>)-butenyl 4-pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; DHAP, dihydroxyacetone 3-phosphate.</p

Isoprene titers and yields from different feeding modules.

<p>Module 1, EMP of strain FMIS 1; Module 2, EDP+PPP of strain FMIS 2; Module 3, EDP of strain FMIS 3, these three strains used glucose as carbon source. Module 4, PPP of strain FMIS 4; Module 5, Dahms pathway of strain FMIS 5, these two strains used D-xylose as carbon source. All strains are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083290#pone.0083290.s003" target="_blank">Table S1</a> in File S1. A 160 mL serum bottle containing 40 mL of semi-defined medium, consisted of M9 salts, 5 g L⁻¹ yeast extract, 10 g L⁻¹ required carbon source and 1 mM thiamine pyrophosphate (TPP), was used for the cultivation of the strains for isoprene production.</p

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered <i>Escherichia coli</i>

<div><p>Embden-Meyerhof pathway (EMP) in tandem with 2-<i>C</i>-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in <i>E. coli</i>. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.</p></div

Aerosol Cross-Linked Crown Ether Diols Melded with Poly(vinyl alcohol) as Specialized Microfibrous Li⁺ Adsorbents

Crown ether (CE)-based Li⁺ adsorbent microfibers (MFs) were successfully fabricated through a combined use of CE diols, electrospinning, and aerosol cross-linking. The 14- to 16-membered CEs, with varied ring subunits and cavity dimensions, have two hydroxyl groups for covalent attachments to poly­(vinyl alcohol) (PVA) as the chosen matrix. The CE diols were blended with PVA and transformed into microfibers via electrospinning, a highly effective technique in minimizing CE loss during MF fabrication. Subsequent aerosol glutaraldehyde (GA) cross-linking of the electrospun CE/PVA MFs stabilized the adsorbents in water. The aerosol technique is highly effective in cross-linking the MFs at short time (5 h) with minimal volume requirement of GA solution (2.4 mL g^–1 MF). GA cross-linking alleviated CE leakage from the fibers as the CEs were securely attached with PVA through covalent CE–GA–PVA linkages. Three types of CE/PVA MFs were fabricated and characterized through Fourier transform infrared-attenuated total reflection, ¹³C cross-polarization magic angle spinning NMR, field emission scanning electron microscope, N₂ adsorption/desorption, and universal testing machine. The MFs exhibited pseudo-second-order rate and Langmuir-type Li⁺ adsorption. At their saturated states, the MFs were able to use 90–99% CEs for 1:1 Li⁺ complexation, suggesting favorability of their microfibrous structures for CE accessibility to Li⁺. The MFs were highly Li⁺-selective in seawater. Neopentyl-bearing CE was most effective in blocking larger monovalents Na⁺ and K⁺, whereas the dibenzo CE was best in discriminating divalents Mg²⁺ and Ca²⁺. Experimental selectivity trends concur with the reaction enthalpies from density functional theory calculations, confirming the influence of CE structures and cavity dimensions in their “size-match” Li⁺ selectivity