Underlying Mechanism of Traditional Herbal Formula Chuang-Ling-Ye in the Treatment of Diabetic Foot Ulcer through Network Pharmacology and Molecular Docking.

BACKGROUND: Chuang-Ling-Ye (CLY) has been clinically proven to be an effective Chinese medicine for the treatment of diabetic foot ulcers (DFU). OBJECTIVES: This study aimed to investigate the possible mechanism of CLY in relation to DFU using network pharmacology and molecular docking. MATERIALS AND METHODS: Firstly, relevant targets of CLY against DFU were obtained from TCMSP, Swiss Target Prediction database and GEO database. Then, topological analysis was employed by Cytoscape to screen the top 6 core active ingredients and the top 8 hub targets. Furthermore, the OmicShare Tools were applied for gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis. Finally, the results of network pharmacology were verified by molecular docking method. RESULTS: CLY has 61 active compounds and 361 targets after de-duplication, and the top 8 hub targets were EGFR, TP53, CCND1, IL-1B, CREBBP, AR, PTGS2 and PGR. GO enrichment analysis is mainly related to signal transducer activity, receptor activity, and molecular transducer activity. KEGG pathway analysis indicated that these shared targets were primarily focused on AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, IL-17 signaling pathway, and JAK-STAT signaling pathway. Molecular docking results showed that physciondiglucoside, 2-cinnamoyl-glucose and kinobeon A were well bound with EGFR, IL-1B, AR and PTGS2. CONCLUSION: This study demonstrated that CLY has anti-oxidative stress and anti-inflammatory effects in the treatment of DFU through various constituents, multiple targets, and multiple pathways, which provides a valuable point of reference for future investigations on CLY.

Preparation and Characterization of a Novel Morphosis of Dextran and Its Derivatization with Polyethyleneimine.

Dextran, a variant of α-glucan with a significant proportion of α-(1,6) bonds, exhibits remarkable solubility in water. Nonetheless, the precipitation of dextran has been observed in injection vials during storage. The present study aimed to establish a technique for generating insoluble dextran and analyze its structural properties. Additionally, the potential for positively ionizing IS-dextran with polyethyleneimine was explored, with the ultimate objective of utilizing IS-dextran-PEI as a promising support for enzyme immobilization. As a result, IS-dextran was obtained by the process of slow evaporation with an average molecular weight of 6555 Da and a yield exceeding 60%. The calculated crystallinity of IS-dextran, which reaches 93.62%, is indicative of its irregular and dense structure, thereby accounting for its water insolubility. Furthermore, positive charge modification of IS-dextran, coupled with the incorporation of epichlorohydrin, resulted in all zeta potentials of IS-dextran-PEIs exceeding 30 mV, making it a promising supporting factor for enzyme immobilization.

Cofactor Metabolic Engineering of Escherichia coli for Aerobic L-Malate Production with Lower CO2 Emissions.

Escherichia coli has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO2 emissions. Here, we aim to address this issue by reducing CO2 emissions of recombinant E. coli during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of E. coli E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of nuoA and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD+ and ATP supplies. The results demonstrate that E23 (ΔnuoA, NOG) exhibited decreased CO2 emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD+ and ATP supply can prompt E. coli to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration.

[Advances in biotransformation of methanol into chemicals].

Methanol has become an attractive substrate for the biomanufacturing industry due to its abundant supply and low cost. The biotransformation of methanol to value-added chemicals using microbial cell factories has the advantages of green process, mild conditions and diversified products. These advantages may expand the product chain based on methanol and alleviate the current problem of biomanufacturing, which is competing with people for food. Elucidating the pathways involving methanol oxidation, formaldehyde assimilation and dissimilation in different natural methylotrophs is essential for subsequent genetic engineering modification, and is more conducive to the construction of novel non-natural methylotrophs. This review discusses the current status of research on methanol metabolic pathways in methylotrophs, and presents recent advances and challenges in natural and synthetic methylotrophs and their applications in methanol bioconversion.

Membrane engineering of Escherichia coli based on "Building bridges" and "Digging tunnels" to improve electro-fermentation of succinate.

Succinate is the end product of anaerobic metabolism of Escherichia coli, and its over-production needs abundant reducing force. Electro-fermentation (EF) is a novel biotechnology to steer and control fermentative processes by supplying extra electrons. However, E.coli is a non-electroactive strain which needs the support of electron shuttle in EF. Here, membrane engineering strategies of "Building bridges" via screening direct electron transport pathway and "Digging tunnels" via screening membrane porins were developed to improve the transmembrane transport of electron during the cathodic electro-fermentation (CEF). As a result, the total electron quantity during electro-fermentation was increased from 1.21 mmol to 7.90 mmol, and succinate yield was increased by 23.3% when these strategies simultaneously were applied to the succinate candidate E. coli Suc260. Hence, this study provides a reference mode for designing and constructing non-electroactive bacteria for electro-fermentation of reductive metabolites.

Identification and characterization of a thermostable GH11 xylanase from Paenibacillus campinasensis NTU-11 and the distinct roles of its carbohydrate-binding domain and linker sequence.

An extracellular thermostable xylanase (XynNTU) from Paenibacillus campinasensis NTU-11, consisted of a glycoside hydrolase (GH) family 11 catalytic domain, a Gly/Pro-rich linker sequence (LS) and a family 6 carbohydrate-binding module (CBM6), was identified and expressed in E. coli BL21. The purified XynNTU had a specific activity of 2750 U/mg and an optimal activity at 60 °C and pH 7.0, and retained a residual activity of 58.4% after incubation (60 °C, 48 h). Two truncated mutants, CBM6-truncated form XynNTU-CDLS, CBM6 and linker-truncated form XynNTU-CD, possessed similar values of optimum pH and temperature as the native XynNTU. XynNTU-CD displayed a lower thermostability than XynNTU, whereas for XynNTU-CDLS, more than 90% of residual activity was remained (60 °C, 48 h), indicating that this enzyme presented a higher thermostability than that of the majority of reported GH11 xylanases. Furthermore, XynNTU and two mutants maintained more than 70% of residual activity at pH values of 5-9. Kinetic measurements suggested that CBM6 had a crucial function in the ability of the enzyme to bind and hydrolyze xylan substrates, while LS had a relatively mild influence. Collectively, a noticeable thermostability and a high specific activity of XynNTU and its truncated form XynNTU-CDLS highlights their potentials for diverse industrial applications.

Low Molar Mass Dextran: One-Step Synthesis With Dextransucrase by Site-Directed Mutagenesis and its Potential of Iron-Loading.

Iron dextran is a common anti-anemia drug, and it requires low molar mass dextran as substrate. In this work, we selected 11 amino acid residues in domain A/B of DSR-MΔ2 within a 5-angstrom distance from sucrose for site-directed mutagenesis by molecular docking. Mutation of Q634 did not affect the enzyme catalytic activity, but showed an obvious impact on the ratio of low molecular weight dextran (L-dextran, 3,000-5,000 Da) and relatively higher molecular weight dextran (H-dextran, around 10,000 Da). L-dextran was the main product synthesized by DSR-MΔ2 Q634A, and its average molecular weight was 3,951 Da with a polydispersity index <1.3. The structural characterization of this homopolysaccharide revealed that it was a dextran, with 86.0% α(1→6) and 14.0% α(1→4) glycosidic linkages. Moreover, L-dextran was oxidized with NaOH and chelated with ferric trichloride, and an OL-dextran-iron complex was synthesized with a high iron-loading potential of 33.5% (w/w). Altogether, mutation of amino acids near the sucrose binding site of dextransucrase can affect the chain elongation process, making it possible to modulate dextran size.

[Bio-based molecules for biosynthesis of nano-metallic materials].

Nano-metallic materials are playing an important role in the application of medicine, catalysis, antibacterial and anti-toxin due to their obvious advantages, including nanocrystalline strengthening effect, high photo-absorptivity, high surface energy and single magnetic region performance. In recent years, with the increasing consumption of global petrochemical resources and the aggravation of environmental pollution, nanomaterials based on bio-based molecules have aroused great concern. Bio-based molecules refer to small molecules and macromolecules directly or indirectly derived from biomass. They usually have good biocompatibility, low toxicity, degradability, wide source and low price. Besides, most bio-based molecules have unique physical, chemical properties and physiological activity, such as optical activity, acid/alkali amphoteric property, hydrophilic property and easy coordination with metal ions. Thus, the corresponding nano-materials based on bio-based molecules also have unique functions, such as anti-inflammatory, anti-cancer, anti-oxidation, antiviral fall blood sugar and blood fat etc. In this paper, we give a comprehensive overview of the preparation and application of nano-metallic materials based on bio-based molecules in recent years.

A staged representation electrochemical stimulated strategy to regulate intracellular reducing power for improving succinate production by Escherichia coli AFP111.

BACKGROUND: Escherichia coli AFP111 was previously engineered for succinate production by eliminating byproducts of synthesis pathways. Still, the succinate yield is limited due to the insufficient NADH supplement, when fed with glucose. Microbial electrolysis cell (MEC) allows microorganisms to perform unbalanced fermentation by establishing polarized cathode interaction. METHODS AND RESULTS: In this study, a cathode electrode was used as an additional electron donor to improve succinate synthesis by E. coli AFP111. In MEC with -0.65 V (vs. Ag/AgCl) poised on cathode electrode, 95.72% electrons were transferred into cells via neutral red (NR), and the ratio of NADH/NAD+ increased by 2.5-fold. Meanwhile, compared with the control experiment, the value of oxidation-reduction potential (ORP) changed from -240 to -265 mV in MEC, which was beneficial for NADH generation. During two-stage fermentation (no potential growth stage followed by electric stimulation) in MEC, succinate yield was increased by 29.09% (the final yield was 0.71 g g-1 ), and glucose consumption rate was enhanced by 36.22%. In addition, the carbon flux was pumped to succinate and pyruvate metabolism was enhanced. CONCLUSION AND IMPLICATIONS: Staged representation of electrochemical stimulated strategy is effective for succinate producing in engineered E. coli by regulating intracellular reducing power, which provides a new concept for producing reduced metabolite in unbalanced fermentation.

Microbial fuel cell-assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes.

BACKGROUND: The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid-tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle. RESULTS: MFC-assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. First, an acid-tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV. CONCLUSIONS: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Comprehensive investigations of 2-phenylethanol production by high 2-phenylethanol tolerating Meyerozyma sp. strain YLG18.

2-Phenylethanol (2-PE) production through bio-synthesis method has become an appealing option owning to the mild conditions and high product selectivity. However, 2-PE is toxic to cells, which is an important limiting factor for the biosynthesis of 2-PE. In this study, a novel 2-PE generating Meyerozyma sp. strain YLG18 was first isolated, which could produce 2-PE through both Ehrlich and Shikimate pathways. Moreover, the indigenous high 2-PE tolerance makes it a promising candidate for high 2-PE production. Response surface methodology and in situ product recovery technology could improve the final 2-PE production to 3.20 g/L, representing the highest 2-PE production by using Meyerozyma sp. Furthermore, genes involved in 2-PE synthesis were identified and their expression levels between Shikimate pathway and Ehrlich pathway were compared. Based on the genomic and transcriptional analysis, a penta-functional enzyme AroM and an aspartate aminotransferase (AAT) with the potential to convert phenylalanine into phenylpyruvate were identified. These findings would help broaden our knowledge and add the pool of known 2-PE generating microbes and genes.

Metabolic engineering of Escherichia coli for L-malate production anaerobically.

BACKGROUND: L-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention. RESULTS: A potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. CONCLUSIONS: The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.

Progress and Prospects of Bioelectrochemical Systems: Electron Transfer and Its Applications in the Microbial Metabolism.

Bioelectrochemical systems are revolutionary new bioengineering technologies which integrate microorganisms or enzymes with the electrochemical method to improve the reducing or oxidizing metabolism. Generally, the bioelectrochemical systems show the processes referring to electrical power generation or achieving the reducing reaction with a certain potential poised by means of electron transfer between the electron acceptor and electron donor. Researchers have focused on the selection and optimization of the electrode materials, design of electrochemical device, and screening of electrochemically active or inactive model microorganisms. Notably, all these means and studies are related to electron transfer: efflux and consumption. Thus, here we introduce the basic concepts of bioelectrochemical systems, and elaborate on the extracellular and intracellular electron transfer, and the hypothetical electron transfer mechanism. Also, intracellular energy generation and coenzyme metabolism along with electron transfer are analyzed. Finally, the applications of bioelectrochemical systems and the prospect of microbial electrochemical technologies are discussed.

Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review.

The diversity of stress responses and survival strategies evolved by microorganism enables them to survive and reproduce in a multitude of harsh environments, whereas the discovery of the underlying resistance genes or mechanisms laid the foundation for the directional enhancement of microbial tolerance to abiotic stresses encountered in industrial applications. Many biological techniques have been developed for improving the stress resistance of industrial microorganisms, which greatly benefited the bacteria on which industrial production is based. This review introduces the main techniques for enhancing the resistance of microorganisms to abiotic stresses, including evolutionary engineering, metabolic engineering, and process engineering, developed in recent years. In addition, we also discuss problems that are still present in this area and offer directions for future research.

Metabolic Regulation of Organic Acid Biosynthesis in Actinobacillus succinogenes.

Actinobacillus succinogenes is one of the most promising strains for succinic acid production; however, the lack of efficient genetic tools for strain modification development hinders its further application. In this study, a markerless knockout method for A. succinogenes using in-frame deletion was first developed. The resulting ΔpflA (encode pyruvate formate lyase 1-activating protein) strain displayed distinctive organic acid synthesis capacity under different cultivation modes. Additional acetate accumulation was observed in the ΔpflA strain relative to that of the wild type under aerobic conditions, indicating that acetate biosynthetic pathway was activated. Importantly, pyruvate was completely converted to lactate under anaerobic fermentation. The transcription analysis and enzyme assay revealed that the expression level and specific activity of lactate dehydrogenase (LDH) were significantly increased. In addition, the mRNA expression level of ldh was nearly increased 85-fold compared to that of the wild-type strain during aerobic-anaerobic dual-phase fermentation, resulting in 43.05 g/L lactate. These results demonstrate that pflA plays an important role in the regulation of C3 flux distribution. The deletion of pflA leads to the improvement of acetic acid production under aerobic conditions and activates lactic acid biosynthesis under anaerobic conditions. This study will help elaborate the mechanism governing organic acid biosynthesis in A. succinogenes.

The Draft Genome Sequence of Methylophilus sp. D22, Capable of Growing Under High Concentration of Methanol.

In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50 g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398 bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.

Recent advances of biofuels and biochemicals production from sustainable resources using co-cultivation systems.

Microbial communities are ubiquitous in nature and exhibit several attractive features, such as sophisticated metabolic capabilities and strong environment robustness. Inspired by the advantages of natural microbial consortia, diverse artificial co-cultivation systems have been metabolically constructed for biofuels, chemicals and natural products production. In these co-cultivation systems, especially genetic engineering ones can reduce the metabolic burden caused by the complex of metabolic pathway through labor division, and improve the target product production significantly. This review summarized the most up-to-dated co-cultivation systems used for biofuels, chemicals and nature products production. In addition, major challenges associated with co-cultivation systems are also presented and discussed for meeting further industrial demands.

Co-production of single cell oil and gluconic acid using oleaginous Cryptococcus podzolicus DSM 27192.

BACKGROUND: The co-production of single cell oil (SCO) with value-added products could improve the economic viability of industrial SCO production. The newly isolated oleaginous yeast Cryptococcus podzolicus DSM 27192 was able to co-produce SCO intracellularly and gluconic acid (GA) extracellularly. In this study, the metabolic regulation of carbon distribution between SCO and GA through process optimization was comprehensively investigated. RESULTS: The carbon flow distribution between SCO and GA was significantly influenced by the cultivation conditions, such as nitrogen sources, glucose concentration and dissolved oxygen concentration. It was found that organic nitrogen sources were beneficial for SCO accumulation, while GA production was decreased. Dissolved oxygen concentration (DOC) was found to enhance SCO accumulation, while high glucose concentration was more favorable for GA accumulation. Hence, a two-stage DOC or glucose concentration-controlled strategy was designed to improve cell growth and direct carbon distribution between SCO and GA. Moreover, C. podzolicus DSM 27192 could degrade its stored lipids to synthesize GA in the late stationary phase, although considerable amounts of glucose remained unconsumed in the culture medium, indicating the importance of fermentation time control in co-production systems. All these observations provide opportunity to favor either the production of SCO or GA or rather their simultaneous production. CONCLUSIONS: Co-production of SCO and GA by C. podzolicus DSM 27192 can improve the economical value for microbial lipid-derived biodiesel production. Moreover, the results of the proposed co-production strategy might give guidance for other co-production systems.

Performance evaluation of a lab-scale moving bed biofilm reactor (MBBR) using polyethylene as support material in the treatment of wastewater contaminated with terephthalic acid.

Untreated terephthalic acid (TPA) wastewaters with high organic loads will cause severe environmental pollution problems. In this study, a lab-scale moving bed biofilm reactor, where biomass of Delftia sp. WL-3 is attached to polypropylene carrier elements, has been tested for TPA bioremediation at 25-27 °C. The system achieved stable operation after a short 15-day start-up period. During the operation period of 65 days, stable chemical oxygen demand (COD) and TPA removal efficiencies of 68% and 76% were maintained with an organic load rate (OLR) and hydraulic retention time of 2.5 kg COD·(m3·d)-1 and 24 h, respectively. In addition, the Scanning Electron Microscope (SEM) showed that high-densities of WL-3 biomass accumulated on the surface of the carrier and formed a rich biofilm, indicating polypropylene carrier can improve the degradation efficiency. On the contrary, the biodegradation ability of stain WL-3 without the polypropylene carrier declined significantly with removal efficiencies of 10% and 15% for COD and TPA. Furthermore, the system exhibited excellent robustness to different OLR and influent matrix ratios, indicating its potential for applications in the treatment of TPA-containment wastewater in the field.