Regulation of intracellular level of ATP and NADH in Escherichia coli to promote succinic acid production / 生物工程学报

Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.

In-Silico Study Of Molecular Properties, Bioactivity And Toxicity Of 2-(Substituted Benzylidene)Succinic Acids And Some Selected Anti-Inflammatory Drugs

Succinic acid and its derivatives have many important uses, especially in pharmaceutical and polymer industry. The 2-(substituted benzylidene)succinic acids also known as substituted phenylitaconic acids are utilized in the synthesis of some lignans, lignanamides and renin inhibitors. In view of this, the present in-silico study aimed to calculate the molecular properties, bioactivity score and toxicity of several benzylidenesuccinic acids as well as some selected anti-inflammatory drugs by computational methods. The study revealed that all the compounds obeyed Lipinski’s rule of five, indicating drug likeness properties. The bioactivity data revealed that the 2-(substituted benzylidene)succinic acids were active as Nuclear receptor ligands, Enzyme inhibitors, GPCR ligands and Ion channel modulators. Among all, 2-(3,5-di-tert-butyl-4-hydroxybenzylidene)succinic acid was predicted as non-toxic with better in-silico molecular properties and bioactivity as Nuclear receptor ligand, Enzyme inhibitor, GPCR ligand, Ion channel modulator and Protease inhibitor compared to some of the predicted anti-inflammatory drugs.

Renal Scarring in Children with Primary Vesicoureteral Reflux Disease

Aims:To determine the incidence of renal scarring among patients with primary vesicoureteral reflux (VUR) and the possible risk factor(s), we studied 69 children (42 girls and 27boys) with VUR attending the Pediatric opd.Study Design:Prospective Observational study.Place and duration of Study:Department of pediatrics,Sher I Kashmir Institute of Medical Sciences,Srinagar, Kashmir between June 2017 to June 2018.Methodology:All the patients were assessed for VUR grade by voiding cystoureterography and for presence of renal scarring by (99 m) technetium dimercapto–succinic acid scintigraphy. Results:Grade of reflux and number of urinary tract infection (UTI) episodes (≥3) were found to be statistically significant risk factors for renal scarring (P <0.05). However,there was no significant association with gender,familial history and laterality of the disease (P >0.05). Similarly, there was no statistically significant difference of frequency of renal scarring among the different age groups (P >0.05).Conclusions:It was concluded that recurrences of UTI and VUR severity are significant risk factors for renal scarring in children with VUR. Therefore,identification of VUR at an early age may offer the opportunity to prevent episodes of UTI and possible formation of renal scars that may result in end-stage renal failure

Isolation and Elucidation of a Novel Isoquinoline Alkaloid from Corydalis hendersonii / 中国实验方剂学杂志

As part of systematic research of Corydalis hendersonii,a typical traditional Tibetan herbal medicine with clearing heat,relieving pain,and lowering blood pressure effects,a novel isoquinoline alkaloid,named hendersine G was isolated from the ethanol extract of the whole plant by various chromatographic techniques,including silica gel column,reverse phase column (ODS),Sephadex LH-20,and semi-preparative HPLC.Its structure was elucidated by MS,NMR and other spectroscopic data analysis.Hendersine G can be regarded as a condensation product of a tetrahydroberberine and a succinic acid,however,its absolute configuration has not been determined due to its structural complexity and less obtained amount.This present study provides an inspiration for further exploration of novel molecules from C.hendersonii.

Control of acetic acid metabolism of recombinant Yarrowia lipolytica for efficient succinic acid production / 生物工程学报

Succinic acid is a high value-added organic acid widely used in food, chemical and pesticide industries. As a new robust non-conventional yeast, Yarrowia lipolytica attracts more and more attention due to its potential for industrial applications. Previously, we obtained a succinic acid-producing strain through gene deletion of succinic acid dehydrogenase subunit encoding gene Ylsdh5, resulting in the strain of PGC01003. However, the recombinant strain produced large amount of acetic acid due to imbalance between glycolysis and TCA cycle which hindered the efficient production of succinic acid. PDH bypass was interfered to decrease the overflow of acetic acid and produce succinic acid under natural pH. Acetic acid was reduced to 4.6 g/L through heterologous expression of acetyl coenzyme A synthase from Salmonella enteric, which was 75.4% of the control strain. Deletion of CoA-transferase gene Ylach1 eliminated acetate formation and improved succinic acid production, and the resulting strain produced as high as 7.0 g/L succinic acid. Our study provides foundation for further construction of efficient cell factory of succinic acid production.

Formulation Optimization of Rutaecarpine Transdermal Patch / 中国药学杂志

OBJECTIVE: To screen pressure sensitive adhesive system and penetration enhancer of rutaecarpine transdermal patch. METHODS: The patch was prepared by solvent evaporation method. The ratio between pressure sensitive adhesive (Eudragit E100), crosslinking agent (succinic acid) and plasticizer (dibutyl sebacate) of the rutaecarpine patch were screened through Box-Behnken design by using adhesion (stick power, shear strength, peel force) as the index. The ratio between pressure sensitive adhesives and chemical enhancers (azone and oleic acid) were screened by adhesion and permeation experiments in vitro using custom design, which were carried out by using improved Franz diffusion cells through excised mice skin. RESULTS: The optimized formulation of rutaecarpine transdermal patch consisted of 83% pressure sensitive adhesives (63.5% Eudragit E100, 5.5% succinic acid, 14% dibutyl sebacate), 10% azone, 6.4% oleic acid and 0.6% rutaecarpine. The stick power, shear strength, and peel force of the patch were 15 steel balls, (10.97 ± 0.32) h and (0.16 ± 0.02) kN•M-1, respectively. The cumulative permeation amount and permeation rate of the patch were (29.71 ± 1.19) μg•cm-2 and (1.36 ± 0.10) μg•cm -2•h -1, respectively. CONCLUSION: The optimized rutaecarpine patch show increased permeation and appropriate adhesion. This study provides the basis for future research.

Chemical constituents from fruits of Forsythia suspense / 中草药

Objective: To study the chemical constituents from the fruits of Forsythia suspensa. Methods: Compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, Sephadex LH-20, and reversed-phase HPLC. Their structures were elucidated by physiochemical property and spectral analysis. Results: Thirteen compounds were isolated and identified as salidroside (1), forsythoside E (2), quinolacetic acid (3), rengyolone (4), senecio lactone (5), cleroindicin C (6), 4-hydroxy-4-isopropylcyclohex-1-enecarboxylic acid (7), oleuropeic acid (8), rel-(1S,2S,4S)-trihydroxy-p-menthane (9), azelaic acid (10), 2-hydroxy-succinic acid 4-methylester (11), glochidioboside (12), and (+)-isolariciresino-9-O-β-D-glucopyranoside (13). Conclusion: Compounds 5-13 are isolated from genus Forsythia Vahl. for the first time.

Determination of Ferrous Succinate Tablets by Ion Chromatography / 中国药师

Objective:To establish a method to determine ferrous succinate in ferrous succinate tablets .Methods:Ion chromatog-raphy was performed on an IonPac AG19(4 mm ×250 mm)anion exchange chromatography column with a guard column of Ion Pac TM AG19( 4 mm ×50 mm);30.00 mmol· L-1 KOH was used as the isocratic eluent at a flow rate of 1.00 ml· min-1 with suppressed conductivity detection ( Curb:75 mA) at the temperature of 30℃.The sample size was 25 μl .Results:The linear range of succinic acid was 2.005 1-25.064 0μg· ml-1(r=0.999 5).The detection limit and quantitation limit was 0.12ng and 0.42ng, respectively. The recovery was 101.45%(RSD=0.43%, n=6).Conclusion:The method is simple, rapid, accurate, reproducible and sensitive , and can be used to determine the ferrous succinate content in ferrous succinate tablets .

Influence of expressing IrrE from Deinococcus radiodurans on osmotic stress tolerance of succinate-producing Escherichia coli / 生物工程学报

Hyper-osmotic stress is one of the key factors that decrease the efficiency of biological succinic acid production. To increase the osmotic stress tolerance of succinate-producing Escherichia coli, we studied the influence of IrrE, an exogenous global regulator, on cell osmotic stress resistance. Fermentation results showed that cell growth and succinic acid production by the recombinant increased under different Na+ concentrations. Meanwhile, the maximum dry cell mass, glucose consumption and succinic acid concentration increased 15.6%, 22% and 23%, respectively, when fermented in a 5-L bioreactor. Expressing IrrE improved cell resistance to hyper-osmotic stress. Further comparison of intracellular osmoprotectants (trehalose and glycerol) concentrations showed that trehalose and glycerol concentrations in the recombinant increased. This suggested that introduction of IrrE could enhance intracellular osmoprotectants accumulation which conferred cell with improved resistance to osmotic stress.

Intoxicación familiar por mercurio elemental: caso clínico / Family poisoned by elemental mercury: a case report

Introduction: mercury is a heavy metal widely dispersed in nature, occurring in three chemical forms. Exposure to mercury at work sites and even at home may be clinically significant. Objective: to update the knowledge about the risks of this toxic element. Case report: the case of a teenager and his family poisoned by elemental mercury is reported. The diagnostic process was difficult, mainly due to an initial presumption of probable infectious etiology, unavailability of key anamnestic data and unusual clinical behavior, with signs and symptoms of multisystem compromise (neurological, hepatic, renal and dermatological compromise). Discussion: the study was based on literature review of various clinical presentations regarding this poisoning and its management, emphasizing the need for dimercaptosuccinic acid chelator. As a major public health problem, the importance of education and implementation of public policies to have a mercury-free environment is discussed.

Introducción: el mercurio es un metal pesado ampliamente distribuido en el medio ambiente, en sus tres formas químicas. La exposición a dicho metal en recintos laborales e incluso en el hogar, puede llegar a ser clínicamente significativa. Objetivo: actualizar el conocimiento acerca de los riesgos de este tóxico. Caso clínico: se presenta el caso clínico de un adolescente y su familia intoxicados por mercurio elemental, cuyo proceso diagnóstico fue difícil, principalmente por la presunción inicial de una probable etiología infecciosa, falta de disponibilidad de datos anamnésticos claves y el inusual comportamiento clínico, con signos y síntomas de compromiso multisistémico (neurológico, hepático, renal y dermatológico). Discusión: se revisa la literatura en relación a las diversas formas de presentación clínica de esta intoxicación y su manejo, destacando la utilidad del quelante ácido dimercaptosuccínico. Por ser un importante problema de salud pública, se destaca la trascendencia de la educación e implementación de políticas públicas por un ambiente libre de mercurio.

Chemical constituents in ethyl acetate extract from aerial part of Gynura divaricata / 中草药

Objective: To study the chemical constituents of ethyl acetate fraction in 80% ethanol reflux extract from the aerial part of Gynura divaricata. Methods: Column chromatographic techniques were applied to isolate and purify the chemical constituents. The chemical structures of the constituents were elucidated on the basis of mass properties and NMR spectral data. Results: Fourteen compounds were isolated and their structures were determined spectroscopically as succinic acid (1), methyl succinate (2), ethyl succinate (3), ethyl methyl succinate (4), 4-hydroxybenzoic acid (5), salicylic acid (6), isovanillic acid (7), p-coumaric acid (8), esculetin (9), quercetin (10), kaempferol (11), isoquercitrin (12), astragalin (13), and rutin (14). Conclusion: Compounds 1-4 and 6-9 are isolated from this plant for the first time.

Chemical constituents from fermented mycelium of Paecilomyces hepiali / 中草药

Objective: To investigate the chemical constituents in the fermented mycelia from Paecilomyces hepiali. Methods: The chemical constituents were isolated and purified by repeated silica gel column, Sephadex LH-20, and semi-preparative HPLC chromatography, and their structures were identified by spectroscopic analyses (MS and NMR). Results: Eleven known compounds were isolated from the ethyl acetate fraction of 95% ethanol extract and were identified as ergosterol D (1), cerevisterol (2), 6-O- methylcerevisterol (3), daucosterol (4), cyclo (L-Leu-L-Trp) (5), cyclo (L-Ile-L-Trp) (6), cyclo (L-Val-L-Trp) (7), 4-[formyl-5- (hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid (8), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid (9), 5-hydroxyl- methylfuroic acid (10), and succinic acid (11). Conclusion: Compounds 1, 2, and 4-9 are isolated from the fungi of Paecilomyces Bain for the first time.

Chemical constituents of Scirpus yagara Ohwi / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Scirpus yagara Ohwi. METHODS: Column chromatography techniques were applied to isolate the constituents. The chemical structures of the constituents were elucidated on the basis of physicochemical properties and spectral data. RESULTS: Forteen compounds were isolated and identified as sanleng diphenyllactone (1), β-sitosterol (2), ferulic acid(3), succinic acid(4), azelaic acid(5), docosanoic acid (6), 6, 7, 10-trihydroxy-8-octadecenoic acid(7), 4, 4-dime-thyl-1, 7-pimelic acid(8), vanillic acid(9), 3, 5-dihydroxy-4-methoxy-benzoic acid(10), p-hydroxybenzaldehyde(II), 3, 4-dihydroxy-benzoic acid(12), 2, 7-dihydroxy xanthone(13) and daucosterol(14). CONCLUSION: Compounds 1-10 and 12-14 are obtained from this plant for the first time, and compounds 1 and 13 were obtained from the genus Scirpus for the first time.

Breeding Actinobacillus succinogenes with Acid-tolerance by Genome Shuffling / 微生物学通报

A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain.After UV-EMS and UV-DES treatments respectively,seven mutated strains with subtle improvements in acid tol-erance were obtained,and were subjected for recursive protoplast fusion.Through three rounds of genome shuffling,four shuffled strains with both higher yield and acid tolerance were obtained.The shuffled strain namely F3-21 could even survive at pH 5.2.The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here.After 48 h of shake-flask fermentation,the succinic acid concentration of F3-21 was 48% higher than that of the parent strain.When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0,the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L,which was increased by 45% compared with that of the parent strain(26.2 g/L).While pH was controlled at 6.5~7.0,the production of succinic acid in 32 h fermentation attained 40.7 g/L.When F3-21 was carried out in fed-batch fermentation,succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation.These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A.succinogenes CGMCC 1593.

The effects of human Na~+-dependent high-affinity dicarboxylate cotransporter 3 and glucose on the transport of succinate in human proximal tubular epithelial cells / 解放军医学杂志

Objective To investigate the effects of Na +-dependent high-affinity dicarboxylate cotransporter 3(NaDC3) and glucose on the transport of succinate in human proximal tubular epithelial cells (HKC) . Methods Western blot was used to detect the expression of hNaDC3 protein in the pcDNA3-hNaDC3, pcDNA3-AhNaDC3 and pcDNA3, transfected HKC, and normal HKC cell. Analysis of succinate contents in the intaking-buffer at the various time points and in the variouse concentrations of glucose were determined by capillary zone electrophoresis. Results The cell transfected with pcDNA3- hNaDC3 was found to have over-expression of hNaDC3 protein for about 1.5 times that of the normal HKC, while succinate content in intaking-buffer was decreased. Although the cells transfected with pcDNA3-AhNaDC3 showed lower expression of hNaDC3 protein, being about 0.6 times that of normal HKC cells, the decrease of succinate in intaking buffer was not obviously effected. There were no differences in hNaDC3 protein content and succinate intaking between the cells transfected with pcDNA3 and normal HKC cells. With the increase in glucose concentration, the decrease of succinate in intaking-buffer was accelerated. Conlusion The results suggested that the overexpression of hNaDC3 protein can cause the succinate-intake more quickly in HKC.

The Progress of Recombinant Escherichia coli for Production of Succinic Acid / 微生物学通报

Succinic acid is regarded as C4 platform chemical, which has a huge potential market with wide applications in biopolymer, food, medicine, and so on. Production of succinic acid by microorganism fer-mentation has attracted much attention in recent times. By fermentation, succinic acid could be produced from renewable resources in place of petroleum with low cost and low pollution; especially CO2 could be fixed by microorganism to form the product, which is a promising way to deal with the green gas. Some E. coli have been chosen as good succinic acid producing strains because of their clear genetic backgrounds, convenience to be modified and controlled, and good growth properties with low nutrients requirements. The progress of genetic engineering strategy and metabolic engineering technology for construction of succinic aicd producing recombinant E. coli has been introduced in this paper with the discussion of the future re-search in this area.

The Application of Ultra High Hydrostatic Pressure in the Screening of Succinic Acid Producing Strain / 微生物学通报

Actinobacillus succinogenes A3 was mutagenized by ultra high hydrostatic pressure for improving the production ability of succinic acid.The effects of pressure,rate of pressure changes and growth periods on the lethality rate of the strain were investigated.The strain in stationary phase was mutagenized at 200 MPa with the pressure changes at the rate of 50 MPa/min.As a result,a mutation strain Actinobacillus succinogenes B19 was obtained,and the concentration of succinic acid could reach 32.2 g/L,which was 17.9%higher than that of the original strain,and in the meanwhile the concentration of acetic acid was decreased by 11.5%.After six generation,the mutant also has good stability of descendiblity for succinic acid production.

A rapid Screening Procedure for Identification of Succinate Producing Anaerobic Strains / 微生物学通报

In order to obtain an efficient screening procedure for identification of succinate producing anaerobic strains,a semi-quantitative paper chromatography method was developed. Lactic acid and acetic acid were identified as the main byproducts in the process of succinate production by the high performance liquid chromatography (HPLC).Succinic acid was completely separated from the byproducts of lactic acid and acetic acid in the same broth developed by paper chromatography.The content of succinic acid was calculated by a semi-quantitative method.The results showed that paper chromatography was a simple and cost effective method that could be utilized to screen anaerobic strains producing succinic acid.

Screening and Identification of a Strain of Actinobacillus succinogenes Producing Succinic Acid by Anaerobic Fermentation / 微生物学通报

A strain,Actinobacillus succinogenes CGMCC 1593,producing succinic acid was isolated from bovin rumen,which could pro- duces succinic acid by anaerobic fermentation.A series of morphological and biochemical characteristics and sequence analysis of 16S rDNA reveal that it belongs to Actinobacillus succinogenes.When cultured anaerobically in medium containing 60 g/L glucose as carbon source,the strain produces 25.8 g/L of succnic acid.

RESEARCH PROCESS IN PRODUCTION OF SUCCINIC ACID BY FERMENTATION / 微生物学通报

Succinic acid is widely used in the field of chemistry and food proces s. Compared to the traditional chemical synthesis, succinic acid production by fermentation has some benefits. The new process with competitive cost uses rene wable agricultural resources including green house gas CO 2 instead of fossil f uels and reduces pollution from chemical synthesis. In this article, research progr e ss in production of succinic acid by fermentation such as microorganisms, metabo lic pathways and its regulation, fermentation and isolation are briefly reviewed .