Wook-Jin Chung

High yield production of D-xylonic acid from D-xylose using engineered Escherichia coli.

An engineered Escherichia coli was constructed to produce D-xylonic acid, one of the top 30 high-value chemicals identified by US Department of Energy. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by disrupting xylose isomerase (XI) and xylulose kinase (XK) genes. The native pathway for xylonic acid catabolism was also blocked by disrupting two genes both encoding xylonic acid dehydratase (yagE and yjhG). Through the introduction of a D-xylose dehydrogenase from Caulobacter crescentus, a D-xylonic acid producing E. coli was constructed. The recombinant E. coli produced up to 39.2 g L(-1) D-xylonic acid from 40 g L(-1) D-xylose in M9 minimal medium. The average productivity was as high as 1.09 g L(-1) h(-1) and no gluconic acid byproduct was produced. These results suggest that the engineered E. coli has a promising application for the industrial-scale production of D-xylonic acid.

Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli.

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. 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.

Dye/water separation through supported liquid membrane extraction

The separation of synthetic dye Rhodamine 6G (R6G) and water was investigated using blended organic liquids in a supported liquid membrane (SLM) extraction system. Liquid membrane (LM) components include octyl alcohol (OcOH) as the dye extractant and a polysiloxane liquid as the stabilizing agent. Initial permeation results revealed the suitability of poly (phenyl methyl) siloxane (PPMS) over poly (octyl methyl) siloxane as the blending agent. The most acceptable condition for dye extraction was determined at feed solution pH congruent with 1, wherein highest distribution coefficient, K(D) (OcOH/H(2)O)=18, was attained. Though permeability decreased at optimal blending condition of 1:1 (w/w) OcOH/PPMS, SLM longevity was exhibited with>98% LM retention after 15 h operation in contrast to pure OcOH SLM system (>60% LM loss). Equilibrium experiments reveal that dye extraction followed Langmuir adsorption principle. The dye transport was elucidated using mass transfer analysis wherein it showed a decrease in overall coefficient (k(o)) at increasing feed concentrations. This was a direct consequence of K(D) decline, which becomes more apparent at higher concentrations when SLM saturation point is approached. At varied hydrodynamic conditions, improved k(o) values were observed up to Re(omega)=10,000 when minimal variation in film resistance is attained. Beyond this condition, k(o) becomes independent from stirring rate effect nonetheless SLM stability is compromised due to shear-induced LM losses.

Phosphorous pentoxide mediated synthesis of 5-HMF in ionic liquid at low temperature

A convenient, mild and environment-friendly dehydration reaction of fructose in ionic liquid using phosphorous pentoxide (P(2)O(5)) has been investigated. The acidic nature of P(2)O(5) along with its hygroscopic properties has been successfully utilized to afford 81.2% yield of 5-hydroxymethylfurfural (5-HMF) at 50°C in 60 mins. Phosphoric acid yielded remarkably less 5-HMF even at higher temperature and longer reaction times. The reaction was optimized by varying different parameters and the results indicated that no rehydration products, such as levulinic acid or formic acid, were formed.

MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli.

BackgroundIsopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization.ResultsIn this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L−1 isopentenol production from 20 g L−1 of glucose.ConclusionThe isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production.

Silver nanoparticles in a polyether - block - polyamide copolymer towards antimicrobial and antifouling membranes

The potential of hydrophilic polyether-block-polyamide copolymer (PEBA) with antimicrobial silver nanoparticles (nano-Ag) to alleviate membrane biofouling was investigated. PEBA solutions of different nano-Ag content were prepared as dense films and as coating materials for ultrafiltration polysulfone (PSf) membranes. Disc diffusion and surface contact tests revealed the capability of the PEBA/nano-Ag films to inhibit the growth of Escherichia coli (E. coli). Contact angle measurements confirmed the hydrophilisation of the PSf surface after coating with PEBA. Field emission scanning electron microscopy, atomic force microscopy (AFM) and fourier transform infrared spectroscopy were performed to confirm the surface modification of PSf. As a proof-of-concept, filtration performances of bare PSf, PEBA coated-and PEBA/nano-Ag coated PSf were compared using a simulated solution inoculated with E. coli as the feed. The results revealed that the hydrophilisation of PSf by coating with PEBA improved the fouling resistance of the membrane as indicated by the retarded flux reduction rate and higher flux recovery. However, PSf exhibited the highest antifouling resistance when coated with PEBA/nano-Ag. The AFM images of used membranes showed that PEBA/nano-Ag minimized the attachment and growth of E. coli on the membrane which abated irreversible biofouling, a problem that was most severe on bare PSf.

Brown algae hydrolysis in 1-n-butyl-3-methylimidazolium chloride with mineral acid catalyst system.

The amenability of three brown algal species, Sargassum fulvellum, Laminaria japonica and Undaria pinnatifida, to hydrolysis were investigated using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride ([BMIM]Cl). Compositional analyses of the brown algae reveal that sufficient amounts of sugars (15.5-29.4 wt.%) can be recovered. Results from hydrolysis experiments show that careful selection of the type of mineral acid as catalyst and control of acid loading could maximize the recovery of sugars. Optimal reaction time and temperature were determined from the kinetic studies on the sequential reducing sugar (TRS) formation and degradation. Optimal reaction times were determined based on the extent of furfurals formation as TRS degradation products. X-ray diffraction and environmental scanning electron microscopy confirmed the suitability of [BMIM]Cl as solvent for the hydrolysis of the three brown algae. Overall results show the potential of brown algae as renewable energy resources for the production of valuable chemicals and biofuels.

A highly selective SBA-15 supported fluorescent “turn-on” sensor for the fluoride anion

In an effort to improve the performance of organic-based F− receptors, organo-silica receptors are being developed taking advantage of the large surface area that mesoporous silica offers. In this work, we investigated the possibility of using a simple “piece-wise” assembly method to immobilize silyl-ether protected fluorescein isothiocyanate (FITC) molecules (receptor 1) on the surface of 3-aminopropyltrimethoxysilane (APTES) and 3-[2-(2-aminoethylamino)ethylamino] propyltrimethoxysilane (APAEAETMS) modified SBA-15 to form sensors ASBA and TSBA, respectively. We showed that aqueous solutions of TSBA (or ASBA) produce distinct changes in absorption and emission spectra upon F− addition due to the F− directed cleavage of Si–O bonds on receptor 1. TSBA (or ASBA) remained stable upon prolonged exposure to UV light (losing ∼0.12% of its fluorescence intensity), and was highly selective towards F− over other common anions (Cl−, Br−, I−, HPO42−, ACO−, and NO3−). Furthermore, the sensitivity of this type of sensor architecture followed a dependence on the kind of amino-silane compound used, which opens up the possibility of synthesizing sensors with tailored detection limits. The detection limit of TSBA and ASBA was 0.02 μM and 0.5 μM, respectively.

Metal-free mild oxidation of 5-hydroxymethylfurfural to 2,5-diformylfuran

The potential of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-hydroxy-TEMPO radical) as an oxidant with [bis(acetoxy)-iodo]benzene (BAIB) and acetic acid (CH3COOH) as co-oxidants to convert 5-hydroxymethylfurfural (5-HMF) into 2,5-diformylfuran (2,5-DFF) was investigated. The effects of oxidant/acid dosages, choice of appropriate solvent, reaction temperature and time were determined to maximize the 2,5-DFF yield. Optimally, 66% 2,5-DFF yield was achieved in TEMPO/BAIB/CH3COOH system at 30 °C after 45 min in ethyl acetate. The reaction system is environmentally benign (metal-free) and energy efficient (mild at short reaction period). With scarce reports on 2,5-DFF production, the developed system provides an alternative route for a better access and wider application of this important platform chemical.

One-Pot Conversion of Carbohydrates into Pyrrole-2-carbaldehydes as Sustainable Platform Chemicals

A practical conversion method of carbohydrates into N-substituted 5-(hydroxymethyl)pyrrole-2-carbaldehydes (pyrralines) was developed by the reaction with primary amines and oxalic acid in DMSO at 90 °C. Further cyclization of the highly functionalized pyrralines afforded the pyrrole-fused poly-heterocyclic compounds as potential intermediates for drugs, food flavors, and functional materials. The mild Maillard variant of carbohydrates and amino esters in heated DMSO with oxalic acid expeditiously produced the pyrrole-2-carbaldehyde skeleton, which can be concisely transformed into the pyrrole alkaloid natural products, 2-benzyl- and 2-methylpyrrolo[1,4]oxazin-3-ones 8 and 9, lobechine 10, and (-)-hanishin 11 in 23-32% overall yields from each carbohydrate.