Synthesis of 3-Phenylserine by a Two-enzyme Cascade System with PLP Cofactor.

A two-enzyme cascade system containing ω-transaminase (ω-TA) and L-threonine aldolase (L-ThA) was reported for the synthesis of 3-Phenylserine starting from benzylamine, and PLP was utilized as the only cofactor in these both two enzymes reaction system. Based on the transamination results, benzylamine was optimized as an advantageous amino donor as confirmed by MD simulation results. This cascade reaction system could not only facilitate the in situ removal of the co-product benzaldehyde, enhancing the economic viability of the reaction, but also establish a novel pathway for synthesizing high-value phenyl-serine derivatives. In our study, nearly 95 % of benzylamine was converted, yielding over 54 % of 3-Phenylserine under the optimized conditions cascade reaction.

Recent Advances of Lignin Functionalization for High-Performance and Advanced Functional Rubber Composites.

The biomass lignin is the only large-volume renewable feedstock that is composed of aromatics but has been largely underutilized and is sought for valorization as a value-added material. Recent research has highlighted lignin as a promising alternative to traditional petrol-based reinforcements and functional additives for rubber composites. This review summarized the recent advances in the functionalization of lignin for a variety of rubber composites, as well as the compounding techniques for effectively dispersing lignin within the rubber matrix. Significant progress has been achieved in the development of high-performance and advanced functional rubber/lignin composites through carefully designing the structure of lignin-based additives and the optimization of interfacial morphologies. This Review discussed the effect of lignin on composite properties, including mechanical reinforcement, dynamic properties, antiaging performance, and oil resistance, and also the advanced stimuli-responsive performance in detail. A critical analysis for the future development of rubber/lignin composites is presented as concluding remarks.

Synthesis and Characterization of Novel Triphenylamine-Containing Electrochromic Polyimides with Benzimidazole Substituents.

Two novel electrochromic aromatic polyimides (named as TPA-BIA-PI and TPA-BIB-PI, respectively) with pendent benzimidazole group were synthesized from 1,2-Diphenyl-N,N'-di-4-aminophenyl-5-amino-benzimidazole and 4-Amino-4'-aminophenyl-4″-1-phenyl-benzimidazolyl-phenyl-aniline with 4,4'-(hexafluoroisopropane) phthalic anhydride (6FDA) via two-step polymerization process, respectively. Then, polyimide films were prepared on ITO-conductive glass by electrostatic spraying, and their electrochromic properties were studied. The results showed that due to the π-π* transitions, the maximum UV-Vis absorption bands of TPA-BIA-PI and TPA-BIB-PI films were located at about 314 nm and 346 nm, respectively. A pair of reversible redox peaks of TPA-BIA-PI and TPA-BIB-PI films that were associated with noticeable color changed from original yellow to dark blue and green were observed in the cyclic voltammetry (CV) test. With increasing voltage, new absorption peaks of TPA-BIA-PI and TPA-BIB-PI films emerged at 755 nm and 762 nm, respectively. The switching/bleaching times of TPA-BIA-PI and TPA-BIB-PI films were 13 s/16 s and 13.9 s/9.5 s, respectively, showing that these polyimides can be used as novel electrochromic materials.

A Hemicyanine-Assembled Upconversion Nanosystem for NIR-Excited Visualization of Carbon Monoxide Bio-Signaling In Vivo.

Carbon monoxide (CO) is considered as the second gasotransmitter involved in a series of physiological and pathological processes. Although a number of organic fluorescent probes have been developed for imaging CO, these probes display excitation within the ultraviolet or visible range, which restrict their applications in the complex biosystems. In the present work, a strategy is developed to construct an upconversion nanoparticles-based nanosystem for upconversion luminescent (UCL) sensing CO. This nanosystem exhibits a fast response to CO with high sensitivity and selectivity in aqueous solution by a near-infrared-excited ratiometric UCL detection method. Meanwhile, laser scanning upconversion luminescence microscope experiments demonstrate that this nanosystem can visualize the endogenous CO bio-signaling in living cells, deep tissues, zebrafish, and living mice by ratiometric UCL imaging. In particular, this nanosystem has been successfully employed in visualization of the endogenous CO bio-signaling through up-regulation of heme oxygenase-1 (HO-1) in the progression of hypoxia, acute inflammation, or ischemic injury. This work demonstrates that the outstanding performance of the nanosystem not only can provide an effective tool for further understanding the role of CO in the physiological and pathological environment, but also may have great potential ability for tracking the expression of HO-1 in living systems.

Reversible Adsorption and Detachment of Saccharomyces cerevisiae on Thermoresponsive Poly(N-isopropylacrylamide)-Grafted Fibers for Continuous Immobilized Fermentation.

Biofilm-mediated continuous fermentation with cells immobilized has gained much attention in recent years. In this study, thermoresponsive poly(N-isopropylacrylamide)-grafted cotton fibers (PNIPAM-CF) were prepared via an improved surface-initiated atom transfer radical polymerization. The modification process imparted switchable wettability to the surface while maintaining the thermal stability and biocompatibility of the CF. During the ethanol transformation, the rapid, reversible cell adsorption and detachment of Saccharomyces cerevisiae were performed through the modulation of wettability, displaying the enhancement of immobilized biomass and immobilization efficiency from 2.20 g/L and 59.43% to 2.81 g/L and 93.32%, respectively. Moreover, the biofilm adsorption matched well with the Freundlich model, indicating that multilayer adhesion was the main mode of biofilm formation. Based on the accumulation of the biofilm, the fabrication and utilization of PNIPAM-CF improved the efficiency of continuous immobilized fermentation, making the ethanol production reach 26.34 g/L in the sixth batch of fermentation. Meanwhile, wettability regulation further enhanced the reusability of the carrier. Therefore, the findings of this study revealed that the application of smart materials in cell immobilization systems had broad prospects for achieving sustainable and continuous catalysis.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production.

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.

Elucidating sludge characteristic, substrate transformation and microbial evolution in a two-sludge denitrifying phosphorus removal system under the impact of HRT.

Granule formation has been recognized as a promising biotechnology in denitrifying phosphorus removal (DPR) systems by facilitating phosphorus accumulation organisms (PAOs) especially denitrifying PAOs (DPAOs), and hydraulic selection made this a more difficult task in continuous operation. This study aimed at exploring the microscopic mechanism and putting forward an effective strategy for DPR granulation under the impact of hydraulic retention time (HRT) (12 h, 10 h, 8 h) in a novel Anaerobic Anoxic Oxic - Moving Bed Biofilm Reactor (A2/O - MBBR) system. With the reduction of intracellular carbon storage (CODintra) efficiency (88.58%-78.53%), nitrogen (N) (85.45%-79.11%) and phosphorus (P) (96.55%-92.47%) removals both dropped, but it exhibited a growth of anoxic phosphorus uptake rate (PURA) (3.79-5.68 mg P/(gMLVSS·h)). The batch tests associating with substrate transformation of poly-ß-hydroxyalkanoates (PHA), glycogen (Gly) agreed well with the corresponding stoichiometry of phosphorus release rate (PRR) (4.83-7.53 mg P/(gMLVSS·h)), PURA (3.55-5.43 mg P/(gMLVSS·h)), oxic phosphorus uptake rate (PURO) (6.08-6.21 mg P/(gMLVSS·h)), and DPAOs/PAOs ratios (57.17%-89.31%), indicating a shift of microbial community. DPR granules gradually stabilized with low sludge volume index (SVI5/SVI30 ratio = 1.1-1.2), dense and compact structure, higher P content (11.63%), more extracted extracellular polymeric substances (EPS) (111.40-160.31 mg/gMLVSS) as proteins/polysaccharides (PN/PS) ratios (1.70-3.47) increased, leading to better sludge settleability and cell hydrophobicity. Fluorescence in situ hybridization (FISH) results showed that PAOs (mainly Cluster I: 20.20%) were the dominant bacteria in the A2/O reactor although a small amount of Defluviicoccus (3.18-3.48%) was responsible for nitrite accumulation, while ammonium-oxidizing bacteria (AOB) (mainly Nitrosomonas: 10.75%) and nitrite-oxidizing bacteria (NOB) (mainly Nitrospira: 15.06%) were enriched in the MBBR.

Roles of nitrate recycling ratio in the A2/O - MBBR denitrifying phosphorus removal system for high-efficient wastewater treatment: Performance comparison, nutrient mechanism and potential evaluation.

The long-term effect of nitrate recycling ratios (R = 100%-500%) on the denitrifying phosphorus removal (DPR) characteristics was studied in a novel two-sludge system, which coupled Anaerobic Anoxic Oxic (A2/O) with Moving Bed Biofilm Reactor (MBBR) for simultaneous nitrogen (N) and phosphorus (P) removals. During the 220 days' operation, effluent COD (30.87-45.15 mg/L) can meet the discharge standard completely, but N and P removals were significantly affected by the R-value, including CODintra removal efficiency (CODintra-Re: 56.09-85.98%), TN removal (TN-Re: 52.06-80.50%), anaerobic PO43- release (PO43--An: 10.66-29.02 mg/L) and oxic PO43- absorption (PO43--O: 2.22-6.26 mg/L). Meanwhile, N and P displayed close correlation with the ΔPO43-/ΔNO3- ratio of 4.20-4.41 at R = 300%-400%, resulting in the high-efficient anoxic poly-ß-hydroxyalkanoates (PHA) utilization (ΔPHAA: 64.88 mgCOD/gVSS). Based on the stoichiometry methodology, at R of 300%-400%, the percentages of phosphorus accumulation organisms (PAOs) and glycogen accumulating organisms (GAOs) contributed to ΔPHAAn (ΔGlyAn) were 71.7%, 28.3% (61.3%, 38.7%) in the anaerobic stage, respectively, while N denitrification rate (NDRA: 3.91-3.93 mg N/(gVSS·h)) and P uptake rate (PURA: 3.76-3.90 mg P/(gVSS·h)) reached the peak, suggesting superior DPR performance with higher contribution of denitrifying PAOs (DPAOs) (70%) than denitrifying GAOs (DGAOs) (30%) in the anoxic stage. Microbial community analysis showed that Accumulibacter (27.66-30.01%) was more enriched than Competibacter (13.41-14.34%) and was responsible for the improved C, N, P removals and DPR characteristics. For optimizing operation, the combined effect of nitrate recycling ratio with other process parameters especially economic evaluation should be considered.

Competitive adsorption of vanillin and syringaldehyde on a macro-mesopore polymeric resin: modeling.

Vanillin and syringaldehyde are widely used as flavoring and fragrance agents in the food products. The potential of a macro-mesoporous adsorption resin was assessed for separation of these binary mixtures. This work focuses on modeling of the competitive adsorption behaviors and exploration of the adsorption mechanism. The characterization results showed the resin had a large BET surface area and specific pore structure with hydrophobic properties. By analysis of the physicochemical properties of the solutes and the resin, the separation mechanism was mainly contributed by hydrophobic effect. Subsequently, the competitive Langmuir isotherm model was used to fit the competitive adsorption isotherms. The pore diffusion coefficient was obtained by macropore diffusion model. Afterwards, a mathematical model was established to predict the breakthrough curves of the binary mixture at various operating conditions. The data and model presented are valuable for design and simulation of the continuous chromatographic separation process.

Towards acetone-uncoupled biofuels production in solventogenic Clostridium through reducing power conservation.

Microbial production of butanol by solventogenic Clostridium has long been complicated with the formation of acetone as an unwanted product, which causes poor product yields and creates a most important problem concerning substrate transformation. Intensive attempts concentrate on carbon conversion pathways to eliminate acetone, but have actually achieved little so far. Here, we believe microbial product distribution can largely depend on how the cell plays its energetic cofactors in central metabolism, and demonstrate that by introducing a synthetic 2,3-butanediol synthesis pathway in Clostridium acetobutylicum as an NADH-compensating module to readjust the reducing power at a systems level, the production of acetone can be selectively and efficiently eliminated (< 0.3 g/L). H2 evolution was reduced by 78%, and the total alcohol yield was strikingly increased by 19% to 0.44 g/g glucose, much higher than those yet reported for butanol fermentation. These findings highlight that it is the loss of reducing power rather than typically manipulated solventogenesis genes that dominates acetone formation. Further study revealed that the NADH-module triggered apparent regulation of pathways involved in electron transfer and reducing power conservation. The study also suggested the key to conservation of intracellular reducing power might essentially lie in the intermediate processes in central metabolism that are related to redox partners, butyrate or C4 branches, and possibly NADH and NADPH specificity. This study represents the first effective redox-based configuration of C. acetobutylicum and provides valuable understandings for redox engineering of native Clostridium species towards advanced production of biofuels and alcohols.

Regulating Cofactor Balance In Vivo with a Synthetic Flavin Analogue.

A novel strategy to regulate cofactor balance in vivo for whole-cell biotransformation using a synthetic flavin analogue is reported. High efficiency, easy operation, and good applicability were observed for this system. Confocal laser scanning microscopy was employed to verify that the synthetic flavin analogue can directly permeate into Escherichia coli cells without modifying the cell membrane. This work provides a promising intracellular redox regulatory approach to construct more efficient cell factories.

Controllable single-photon nonreciprocal propagation between two waveguides chirally coupled to a quantum emitter.

We investigate coherent controlling single-photon nonreciprocal propagation in a pair of waveguides chirally coupled to an atom by using a classical optical field. The results show that for a nonresonant photon, the perfect single-photon nonreciprocal propagation can be realized by adjusting the Rabi frequency and detuning. Furthermore, the nonreciprocal propagation is switchable by using the classic field. The calculated results also show that the system can be used as a frequency filter to filter out some special frequencies for single-photon nonreciprocal propagation. The influences of nonperfect chiral coupling and dissipations on the nonreciprocal propagation are also shown.

Screening of promoters from Arthrobacter sp. CGMCC 3584 using a green fluorescent protein reporter system.

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6-hydroxyl-D-nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.

Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum.

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum.

Redirecting metabolic flux in Saccharomyces cerevisiae through regulation of cofactors in UMP production.

Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. To understand the impact of cofactors on physiological functions, a systematic approach was applied, which involved redox state analysis, energy charge analysis, and metabolite analysis. Using uridine 5'-monophosphate metabolism in Saccharomyces cerevisiae as a model, we demonstrated that regulation of intracellular the ratio of NADPH to NADP(+) not only redistributed the carbon flux between the glycolytic and pentose phosphate pathways, but also regulated the redox state of NAD(H), resulting in a significant change of ATP, and a significantly altered spectrum of metabolic products.

Control of glycolytic flux in directed biosynthesis of uridine-phosphoryl compounds through the manipulation of ATP availability.

Adenosine triphosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in control of metabolic flux. Considering the importance of ATP in regulation of the glycolytic pathway, the use of ATP-oriented manipulation is a rational and efficient route to regulate metabolic flux. In this paper, a series of efficient ATP-oriented regulation methods, such as changing ambient temperature and altering reduced nicotinamide adenine dinucleotide (NADH), was developed. To satisfy the different demand for ATP at different phases in directed biosynthesis of uridine-phosphoryl compounds, a multiphase ATP supply regulation strategy was also used to enhance to yield of target metabolites.

A hemicyanine-modified upconversion nanoprobe for NIR-excited evaluating superoxide signaling in drug-induced liver injury.

BACKGROUND: Drug-induced liver injury (DILI) is the most important standard for the entrance of clinical drugs into the pharmaceutical market. The elevation of superoxide anion (O2•-) during drug metabolism can mediate apoptosis of hepatocytes and further generation of liver damage. Therefore, developing an effective imaging method for evaluating O2•- levels during DILI is of great importance. However, current reported O2•- fluorescent probes either use short excitation wavelengths or a single intensity detection system, limiting the accurate quantification of O2•- in deep tissue in vivo. RESULTS: We developed a NIR-excited ratiometric nanoprobe (CyD-UCNPs) by assembly of O2•--sensitive hemicyanine dyes (CyD) on the surface of Tm/Er-codoped upconversion nanoparticles (UCNPs) with the assistance of α-cyclodextrin, which exhibited a robust "turn-on" ratiometric sensing signal. In vitro experiments indicated that CyD-UCNPs respond well to O2•- with high selectivity. Furthermore, by taking advantage of the outstanding optical properties produced by the luminescent resonance energy transfer between the UCNPs and CyD upon the excitation of 980 nm, the ratiometric upconversion luminescence signal of CyD-UCNPs was successfully utilized to monitor the fluctuation of O2•- levels under phorbol-12-myristate-13-acetate (PMA)/cisplatin-induced oxidative stress in living cells, liver tissues, and zebrafish. More importantly, endogenous change in O2•- levels in the liver sites of mice during DILI and its prevention with L-carnitine was visualized using CyD-UCNPs. SIGNIFICANCE: This study provides a ratiometric NIR-excited imaging strategy for investigating the correlation between O2•- levels and DILI and its prevention, which is significant for early diagnosis of DILI and preclinical screening of anti-hepatotoxic drugs in vivo.

NIR-excited imaging of drug-induced liver injury using a superoxide-activated ratiometric upconversion luminescence nanoprobe.

Drug-induced liver injury (DILI) poses a significant risk to human health. Increasing evidence indicates that the superoxide anion (O2•-), as the precursor of the other reactive oxygen species, is key in the pathological processes associated with DILI. Nonetheless, understanding of the mechanisms of DILI is difficult due to the lack of an imaging tool for monitoring the fluctuation of O2•- levels during the progression of DILI. Herein, we developed an upconversion nanoprobe (Rbh-UCNs) for in vivo ratiometric tracking of endogenous O2•- in DILI. In this design, the addition of O2•- triggers the luminescent resonance energy transfer between Rbh and UCNs, which significantly enhances absorption centered at 534 nm and translates into a distinct decrease of the UCL emission at 543 nm, while the UCL emission peak at 654 nm and 800 nm are not significantly affected, offering a ratiometric UCL signal for the quantitative detection of O2•-. In addition, Rbh-UCNs could effectively visualize endogenous O2•- in living cells, zebrafish, and liver tissues upon stimulation with PMA or cisplatin. More importantly, tissue imaging of the liver region of mice revealed that the fluctuation of O2•- levels is associated with DILI and the protective effect of L-carnitine against DILI. Altogether, this study provides an available method for a deeper comprehension of the mechanisms underlying DILI and accelerating the development process of hepatoprotective medicines.

Dopamine-assisted surface functionalization of saccharide-responsive fibers for the controlled harvesting and continuous fermentation of Saccharomyces cerevisiae.

Continuous fermentation processes increasingly emphasized cell recycling, utilization, and renewal. In this study, to improve the sustainability of the immobilized Saccharomyces cerevisiae, the cells were recovered on the surface of the glucose-responsive supports through manipulating the competitive interactions of phenylboric acid groups between glycoproteins on the cells and glucose. Through a dopamine (DA)-assisted deposition approach, 3-acrylamidophenylboronic acid (APBA) was integrated to design the saccharide-sensitive cotton fibers (APBA@PDA-CF). The optimal co-deposition time (5 h) and ratio (1:1) resulted in an impressive immobilization efficiency of 69.64%. Meanwhile, 93.23% of Saccharomyces cerevisiae was captured and harvested on the surface of APBA@PDA-CF with the fermentation course through regulating the competitive interactions of phenylboric acid groups between glycoproteins on the cells and glucose regardless of pH. Notably, a strong interaction between the yeast cells and APBA@PDA-CF was observed at a low glucose concentration (0.1～2 g/L), with reduced sensitivity at high glucose concentrations (>5 g/L). Moreover, the ethanol production and yield could be increased to 25.37 g/L and 42.4% in the fifth-batch fermentation, respectively. Therefore, based on the feasible and versatile co-deposition method, this study not only broadened the application scope of APBA, but also explored the broad prospects of smart materials in cell immobilization, recovery and continuous fermentation.

Nonactivated Aziridine Synthesis by Intermolecular Polarity-Mismatched Carboamination of Unactivated Alkenes with Unactivated Alkyl Halides.

A general intermolecular polarity-mismatched carboamination reaction of unactivated alkenes with unactivated alkyl halides has been developed. A series of nonactivated alkyl-substituted aziridines were constructed in exclusive regioselectivity. The dual polarity-mismatched mechanism might be involved.