A novel co-production of cadaverine and succinic acid based on a thermal switch system in recombinant Escherichia coli.

BACKGROUND: Polyamide (nylon) is an important material, which has aroused plenty of attention from all aspects. PA 5.4 is one kind of nylon with excellent property, which consists of cadaverine and succinic acid. Due to the environmental pollution, bio-production of cadaverine and succinic acid has been more attractive due to the less pollution and environmental friendliness. Microbes, like Escherichia coli, has been employed as cell factory to produce cadaverine and succinic acid. However, the accumulation of cadaverine will cause severe damage on cells resulting in inhibition on cell growth and cadaverine production. Herein, a novel two stage co-production of succinic acid and cadaverine was designed based on an efficient thermos-regulated switch to avoid the inhibitory brought by cadaverine. RESULTS: The fermentation process was divided into two phase, one for cell growth and lysine production and the other for cadaverine and succinic acid synthesis. The genes of ldhA and ackA were deleted to construct succinic acid pathway in cadaverine producer strain. Then, a thermal switch system based on pR/pL promoter and CI857 was established and optimized. The fermentation conditions were investigated that the optimal temperature for the first stage was determined as 33 â„ƒ and the optimal temperature for the second stage was 39 â„ƒ. Additionally, the time to shifting temperature was identified as the fermentation anaphase. For further enhance cadaverine and succinic acid production, a scale-up fermentation in 5 L bioreactor was operated. As a result, the titer, yield and productivity of cadaverine was 55.58 g/L, 0.38 g/g glucose and 1.74 g/(L·h), respectively. 28.39 g/L of succinic acid was also obtained with yield of 0.19 g/g glucose. CONCLUSION: The succinic acid metabolic pathway was constructed into cadaverine producer strain to realize the co-production of succinic acid and cadaverine. This study provided a novel craft for industrial co-production of cadaverine and succinic acid.

Enhancing catalytic stability and cadaverine tolerance by whole-cell immobilization and the addition of cell protectant during cadaverine production.

A whole-cell (cadaverine-producing strain, Escherichia coli AST3) immobilization method was developed for improving catalytic activity and cadaverine tolerance during cadaverine production. Cell-immobilized beads were prepared by polyvinyl alcohol (PVA) and sodium alginate (SA) based on their advantages in biocatalyst activity recovery and mechanical strength. The following optimal immobilization conditions were established using response surface methodology: 3.62% SA, 4.71% PVA, 4.21% CaCl2, calcification, 12 h, and freezing for 16 h at - 80 °C, with a cell concentration of 0.3% (g dry cell weight (DCW) per 100 mL) of immobilized beads. After a 2-h bioconversion, the immobilized beads maintained 85% of their original biocatalyst activity, which was 1.8-fold higher than that of free cells. Furthermore, the effects of cell protectants on immobilized biocatalyst activity were examined by fed-batch bioconversion experiments. The results showed that the addition of polyvinylpyrrolidone (PVP) into the immobilized matrix effectively protected biocatalyst activity, with 95% of the relative activity remaining after the 2-h bioconversion. The performance of PVA-SA-PVP-immobilized E. coli AST3 showed continuous production of cadaverine, with an average cadaverine yield of 29 ± 1 g gDCW-1 h-1 after 12 h, suggesting that this method is capable of industrial scale cadaverine production.

Enhancement of cellulose degradation in freshwater sediments by a sediment microbial fuel cell.

OBJECTIVE: To demonstrate that an enhanced sediment microbial fuel cell (SMFC) system can accelerate the degradation of cellulose in fresh water sediments as the accumulation of cellulose in lake sediments may aggravate the lake marsh, increase organic matter content and result in rapid deterioration of water quality and damage the ecosystem. RESULTS: After 330 days the highest cellulose removal efficiency (72.7 ± 2.1 %) was achieved in the presence of a SMFC with a carbon nanotube decorated cathode, followed by a SMFC without the cathode decoration (64.4 ± 2.8 %). The lowest cellulose removal efficiency (47.9 ± 2.1 %) was in the absence of SMFC. The sediment characterization analysis confirmed that the carbon nanotube decorated cathode enhances the electron transfer rate in the SMFC and improves the dissolved organic matter oxidation rate. CONCLUSION: This study offers a relatively simple and promising new method for cellulose degradation in sediment.

Enhanced oral bioavailability of lurasidone by self-nanoemulsifying drug delivery system in fasted state.

OBJECTIVE: The purpose of this work was to develop a new formulation to enhance the bioavailability and reduce the food effect of lurasidone using self-nanoemulsifying drug delivery systems (SNEDDSs). METHODS: The formulation of lurasidone-SNEDDS was selected by the solubility and pseudo-ternary phase diagram studies. The prepared lurasidone-SNEDDS formulations were characterized for self-emulsification time, effect of pH and robustness to dilution, droplet size analysis, zeta potential and in vitro drug release. Lurasidone-SNEDDSs were administered to beagle dogs in fed and fasted state and their pharmacokinetics were compared to commercial available tablet as a control. RESULTS: The result showed lurasidone-SNEDDS was successfully prepared using Capmul MCM, Tween 80 and glycerol as oil phase, surfactant and co-surfactant, respectively. In vitro drug release studies indicated that the lurasidone-SNEDDS showed improved drug release profiles and the release behavior was not affected by the medium pH with total drug release of over 90% within 5 min. Pharmacokinetic study showed that the AUC(0-∞) and Cmax for lurasidone-SNEDDS are similar in the fasted and fed state, indicating essentially there is no food effect on the drug absorption. CONCLUSION: It was concluded that enhanced bioavailability and no food effect of lurasidone had been achieved by using SNEDDS.

Comparison of the hydrodynamics and mass transfer characteristics in internal-loop airlift bioreactors utilizing either a novel membrane-tube sparger or perforated plate sparger.

Two gas spargers, a novel membrane-tube sparger and a perforated plate sparger, were compared in terms of hydrodynamics and mass transfer (or oxygen transfer) performance in an internal-loop airlift bioreactor. The overall gas holdup ε T, downcomer liquid velocity V d, and volumetric mass transfer coefficient K L a were examined depending on superficial gas velocity U G increased in Newtonian and non-Newtonian fluids for the both spargers. Compared with the perforated plate sparger, the bioreactor with the membrane-tube sparger increased the values of ε T by 4.9-48.8% in air-water system when the U G was from 0.004 to 0.04 m/s, and by 65.1-512.6% in air-CMC solution system. The V d value for the membrane-tube sparger was improved by 40.0-86.3%. The value of K L a was increased by 52.8-84.4% in air-water system, and by 63.3-836.3% in air-CMC solution system. Empirical correlations of ε T, V d, and K L a were proposed, and well corresponding with the experimental data with the deviation of 10%.

Isolation of mono-caffeoylquinic acids from tobacco waste using continuous resin-based pre-separation and preparative HPLC.

Three isomers of mono-caffeoylquinic acid, specifically, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid, were successfully isolated from a crude extract of tobacco (Nicotiana tobaccum L.) wastes using continuous resin-based pre-separation and preparative high-performance liquid chromatography (HPLC). The extract of tobacco wastes was continuously pre-separated by resin-based columns packed with D101 and XAD-4, yielding total mono-caffeoylquinic acids with a purity of 67.71% and a recovery rate of 90.06%. Variables affecting resolution and productivity of three mono-caffeoylquinic acid isomers in preparative HPLC (i.e. mobile-phase composition, pH, flow rate and loading amount) were studied. The optimum chromatographic conditions were determined to be a mobile phase consisting of 15% (v/v) methanol and aqueous acetic acid with a pH of 4.5, a flow rate of 4.0 mL/min, a loading amount of 4 mL and a detection wavelength of 360 nm. From 300 mg of loading sample, 56.3 mg of 3-O-caffeoylquinic acid, 92.8 mg of 5-O-caffeoylquinic acid and 73.1 mg of 4-O-caffeoylquinic acid were obtained in a single run, each with a purity of over 98% by HPLC. The structures of the isolated compounds were elucidated by ESI-MS, (1) H-NMR and (13) C-NMR spectral data.

Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation.

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l(-1) of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g(-1). When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l(-1) of ABE with a yield of 0.34 g g(-1), including 2.2 g l(-1) acetone, 6.8 g l(-1) butanol, and 0.5 g l(-1) ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.

Dissolution of microcrystalline cellulose in phosphoric acid--molecular changes and kinetics.

In this study, we aimed to dissolve microcrystalline cellulose (MCC) with phosphoric acid to obtain high-quality fermentable saccharides. MCC was directly dissolved in phosphoric acid (the concentration was 83%) for 10 hours at temperatures of 30, 50, and 70 degrees C. The structural changes of MCC were determined in detail with X-ray powder diffraction, solid-state cross-polarization magic angle spinning (13)C-NMR, and X-ray photoelectron spectroscopy. The kinetics of MCC decrystallization during treatment with phosphoric acid was also compared at 30, 50, and 70 degrees C. With the assumption of first order kinetics, the Arrhenius parameters of K, A(0) and E(a) were calculated. The rate constants of decrystallization reaction (K) were 0.06, 0.17, and 0.12 h(-1) respectively. The pre-exponential factor (A(0)) was 1.2 x 10(6) h(-1), and the activation energy (E(a)) was 42.4 kJ/mol.

High-yield production of D-1,2,4-butanetriol from lignocellulose-derived xylose by using a synthetic enzyme cascade in a cell-free system.

Approaches using metabolic engineering to produce D-1, 2, 4-butanetriol (BT) from renewable biomass in microbial systems have achieved initial success. However, due to the lack of incomplete understanding of the complex branch pathway, the efficient fermentation system for BT production was difficult to develop. Here we reconstituted a cell-free system in vitro using purified enzymes to produce BT from d-xylose. The factors that influencing the efficiency of cell-free system, including enzyme concentration, reaction buffer, pH, temperature, metal ion additives and cofactors were first identified to define optimal reaction conditions and essential components for the cascade reaction. Meanwhile, a natural cofactor recycling system was found in cell-free system. Finally, we were able to convert 18 g/L xylose to 6.1 g/L BT within 40 h with a yield of 48.0%. The feasibility of cell-free system to produce BT in corncob hydrolysates was also determined.

Surface-assisted assembly of a histidine-rich lipidated peptide for simultaneous exfoliation of graphite and functionalization of graphene nanosheets.

Biological molecules have promising potential to exfoliate graphite and produce biocompatible graphene nano-materials for biomedical applications. Here, a systematic design of a histidine-rich lipidated peptide sequence is presented that simultaneously exfoliates graphite flakes and functionalizes the resulting graphene nanosheets (∼150 nm lateral size) with long-term dispersion stability in aqueous solution (>8 months). The details of peptide/peptide and peptide/graphite interactions are probed using various microscopy, spectroscopy and molecular dynamics simulation methods. The results show that histidine and stearic acid interact with the graphite surface through π-π stacking and hydrophobic forces, respectively. Surface-assisted assembly of peptide molecules is then initiated via hydrogen bonds between deprotonated histidine segments, and a textured peptide nano-structure is formed. The work of adhesion between the peptide and graphite is found to be high enough to promote exfoliation of graphite flakes through layer-by-layer peeling of graphene nanosheets. The positively charged arginine in the peptide is exposed outward, and is responsible for the stable dispersion. The peptide molecules are sufficiently small, presenting the possibility to insert into and increase the spacing between the graphitic layers for enhanced exfoliation. The peptide-functionalized graphene nanosheets not only show great biocompatibility with cells in vitro, but also enhance cancer drug uptake by the cells.

Superior anti-neoplastic activities of triacontanol-PEG conjugate: synthesis, characterization and biological evaluations.

Triacontanol (TA, C30H62O), abundantly present in plant cuticle waxes and bee waxes, has been found to display promising anti-neoplastic potentials. As a long chain fatty alcohol, TA possesses limited aqueous solubility, which hinders its medicinal application. To overcome its solubility barrier, a polymer prodrug was synthesized through attaching TA to poly ethylene glycol (PEG), using succinic acid as a linker with bifunctional amide and ester bonds. Anti-neoplastic effects of PEG-TA were assessed in LoVo and MCF7 cells, anti-proliferative and apoptosis-inducing activities were subsequently confirmed in mouse xenograft model. Encouragingly, PEG-TA possessed selective anti-cancer ability. It did not exhibit significant cytotoxicity on normal cells. Mechanistic examination revealed inhibition of NF-κB nuclear translocation, suppression on matrix degradation enzyme and down-regulation of angiogenic signaling might contribute to its anti-malignant effects. Pharmacokinetics clearly indicated PEGylated TA (named as mPEG2K-SA-TA) substantially enhanced TA delivery with increased plasma exposure (19,791 vs. 336.25 ng·mL-1·h-1, p < .001), mean residence time (8.46 vs. 2.95 h, p < .001) and elimination half-life (7.78 vs. 2.57 h, p < .001) compared to those of original TA. Moreover, mPEG2K-SA-TA appeared to be safe in preliminary toxicological assessment. PEGylated TA also emerged as a functional carrier to deliver hydrophobic chemotherapeutic agents, since it readily self-assembled to micelles in aqueous solution with a low critical micelle concentration (CMC, 19.1 µg·mL-1). Conclusively, PEG-TA conjugate displayed superior anti-neoplastic activities and low toxicity, as well as facilitated the delivery of other hydrophobic agents, which appeared to be an innovative strategy for cancer therapy.

PEGylated Triacontanol Substantially Enhanced the Pharmacokinetics of Triacontanol in Rats.

Triacontanol (TA), a natural compound with various health benefits, is extensively used as a nutritional supplement. The therapeutic and nutraceutical applications of TA are limited due to its poor aqueous solubility. PEGylated triacontanol (PEGylated TA) was designed to improve the solubility and pharmacokinetics of TA. After PEGylation, the solubility (∼250 g·L-1 versus 9 × 10-14 g·L-1), body residence (MRT, 9.40 ± 2.03 h versus 2.59 ± 0.705 h, p < 0.001), and systemic exposure (AUC0-inf, 29.1 ± 5.33 µM·h versus 0.529 ± 0.248 µM·h, p < 0.001) of TA were all significantly increased compared to pristine TA. When intravenously administered (6.85, 22.8, and 68.5 µmol·kg-1) in rats, PEGylated TA exhibited a slow clearance (44.8 ± 8.62, 47.9 ± 5.18, and 46.9 ± 16.5 mL·h-1·kg-1), long elimination half-life (8.76 ± 0.96, 10.4 ± 1.66, and 11.1 ± 2.81 h), and abundant systemic exposure (AUC0- t, 155 ± 24.2, 523 ± 56.2, and 1709 ± 245 µM·h). Meanwhile, its metabolite TA showed a high AUC0- t (28.4 ± 5.14, 151 ± 25.4, and 797 ± 184 µM·h) and slow elimination ( t1/2, 10.1 ± 2.03, 7.78 ± 1.74, and 6.82 ± 0.58 h). Our results demonstrated that PEGylated TA has superior pharmacokinetics, which enhanced its nutritional and pharmacodynamic potency, and thus warrants further investigations.

Design and Characterization of a Multifunctional pH-Triggered Peptide C8 for Selective Anticancer Activity.

Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic ß-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications.

Succinate production by metabolically engineered Escherichia coli using sugarcane bagasse hydrolysate as the carbon source.

Efficient biosynthesis of succinate from a renewable biomass resource by engineered Escherichia coli is reported in this paper. Fermentation of sugarcane bagasse hydrolysate by engineered E. coli BA204, a pflB, ldhA, and ppc deletion strain overexpressing the ATP-forming phosphoenolpyruvate carboxykinase from Bacillus subtilis 168, produced a final succinate concentration of 15.85 g L(-1) with a high yield of 0.89 g L(-1) total sugar under anaerobic conditions. During dual-phase fermentations, initial aerobic growth facilitated subsequent anaerobic succinate production, with a final succinate concentration of 18.88 g L(-1) and a yield of 0.96 g g(-1) total sugar after 24 h of anaerobic fermentation. The high succinate yield from sugarcane bagasse hydrolysate demonstrated a great potential application of renewable biomass as a feedstock for the economical production of succinate using metabolically engineered E. coli.

Efficient succinic acid production from lignocellulosic biomass by simultaneous utilization of glucose and xylose in engineered Escherichia coli.

To enhance succinic acid formation during xylose fermentation in Escherichia coli, overexpression of ATP-forming phosphoenolpyruvate carboxykinase (PEPCK) from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production. However, BA204 displays a low yield of glucose fermentation and sequential glucose-xylose utilization under regulation by the phosphotransferase system (PTS). To improve the capability of glucose fermentation and simultaneously consume sugar mixture for succinic acid production, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming PEPCK, named E. coli BA305, was constructed. As a result, after 120 h fed-batch fermentation of sugarcane bagasse hydrolysate, the dry cell weight and succinic acid concentration in BA305 were 4.58 g L(-1) and 39.3 g L(-1), respectively.

Repetitive succinic acid production from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered Escherichia coli.

In this study, repetitive production of succinic acid from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered E. coli is reported. Escherichia coli BA305, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming phosphoenolpyruvate (PEP) carboxykinase (PEPCK), produced a final succinic acid concentration of 83 g L(-1) with a high yield of 0.87 g g(-1) total sugar in 36 h of three repetitive fermentations of sugarcane bagasse hydrolysate. Furthermore, simultaneous consumption of glucose and xylose was achieved, and the specific productivity and yield of succinic acid were almost maintained constant during the repetitive fermentations.

Propionic acid production in a plant fibrous-bed bioreactor with immobilized Propionibacterium freudenreichii CCTCC M207015.

A plant fibrous-bed bioreactor (PFB) was constructed for propionic acid production. Sugar cane bagasse was applied to the PFB as immobilizing material. Starting at a concentration of 80g/L of glucose, Propionibacterium freudenreichii CCTCC M207015 produced 41.20±2.03g/L of propionic acid at 108h in the PFB. The value was 21.07% higher than that produced by free cell fermentation. Intermittent and constant fed-batch fermentations were performed in the PFB to optimize the fermentation results. The highest propionic acid concentration obtained from constant fed-batch fermentation was 136.23±6.77g/L, which is 1.40 times higher than the highest concentration (97.00g/L) previously reported. Scanning electron microscopy analysis showed that cells exhibited striking changes in morphology after PFB domestication. Compared with free cell fermentation, the fluxes of propionic acid synthesis and the pentose phosphate pathway in PFB fermentation increased by 84.65% and 227.62%, respectively. On the other hand, a decrease in succinic and acetic acid fluxes was also observed. The metabolic flux distributions of the two PFB fed-batch fermentation strategies also demonstrated that constant fed-batch fermentation is a more beneficial method for the immobilized production of propionic acid. The relevant key enzyme activities and metabolic flux variations of the batch cultures showed good consistency. These results suggest that the PFB was effective in high-concentration propionic acid production.

Co-immobilization mechanism of cellulase and xylanase on a reversibly soluble polymer.

Cellulase and xylanase from Trichoderma reesei were immobilized simultaneously on Eudragit L-100, a reversibly soluble polymer. The effects of polymer concentration and polymer precipitation pH on enzyme activity recovery were investigated at an enzyme complex concentration of 1%. The immobilization mechanism of cellulase and xylanase on the polymer was discussed. An activity recovery of 75% and 59% was obtained for the cellulase and the xylanase, respectively, under the condition of a polymer concentration at 2% and a polymer precipitation pH at 4.0. Most zymoproteins might be connected to the polymer by electrostatic attraction in a medium of pH 4.8. In addition, the covalent coupling between the zymoproteins and the polymer was demonstrated by the infrared spectrograms. It was suggested that dehydration-condensation reaction occurred between the zymoproteins and the polymer during the immobilization.

Enhancing the enzymatic hydrolysis of corn stover by an integrated wet-milling and alkali pretreatment.

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.

Effect of phosphoric acid pretreatment on enzymatic hydrolysis of microcrystalline cellulose.

Microcrystalline cellulose (MCC) was pretreated with phosphoric acid at 323K for 10h. X-ray diffraction (XRD) and Atomic Force Microscope (AFM) analyses revealed that the fiber surface morphology of pretreated MCC (P-MCC) were uneven and rough with the crystalline diffraction peaks of P-MCC decreased to a distinct range. The X-ray Photoelectron Spectroscopy (XPS) analysis showed that the uneven and rough surface of P-MCC could enhance the adsorption of cellulose to the molecular surface of cellulose, which is one of the key factors affecting enzymatic hydrolysis of cellulose. A reversible first order kinetics was employed to describe the adsorption kinetics of cellulase to MCC and P-MCC, and the adsorption rate constants of MCC and P-MCC were found to be 0.016, 0.024, 0.041, and 0.095, 0.149, 0.218min(-1), respectively at 278K, 293K and 308K. The activation energies of MCC and P-MCC hydrolysis reactions were found to be 22.257 and 19.721kJ mol(-1). The major hydrolysis products of MCC and P-MCC were cellobiose and glucose. Hydrolysis of MCC for 120h resulted in yields of glucose (7.21%), cellobiose (13.16%) and total sugars (20.37%). However, after the pretreatment with phosphoric acid, the corresponding sugar yields resulted from enzymatic hydrolysis of P-MCC were increased to 24.10%, 41.42%, and 65.52%; respectively, which were 3.34, 3.15, and 3.22 times of the sugars yields from enzymatic hydrolysis of MCC.