Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid / 生物工程学报

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Efficient cascade biosynthesis of (S)-2-hydroxybutyric acid / 生物工程学报

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum / 生物工程学报

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.

Effect of key notes of TCA cycle on L-glutamate production / 生物工程学报

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Analysis of risk factors of pressure ulcer healing / 中国实用护理杂志

Objective To investigate the risk factors of wound healing 30 days after treatment in patients with pressure ulcer. Methods Two hundred and forty-five hospitalized patients with pressure ulcer between January 2015 and October 2016 were selected. They were divided into the healed group and the unhealed group 30 days after the treatment. The influencing factors 13 indexes including age, gender, edema, the number of diseases, the number of pressure ulcer, the size of pressure ulcer, PUSH score (Pressure Ulcer Scale Healing), hemoglobin, serum albumin, low density lipoprotein cholesterol and blood homocysteine underwent single factor and Logistic regression analysis. Results Single factor analysis of 12 indicators of risk factors affecting healing were age, Hb, Alb, Hcy, area of ulcer and PUSH scores of the 6 indicators in the regression equation (Z=3.11-6.63, All P < 0.01), gender also entered the regression equation (χ2=0.19, P<0.01);further multivariate regression analysis showed that there were statistically significant differences in PUSH score index (χ2=10.47, P<0.01, OR=0.001, 95%confidence interval were 0.001-0.037). Conclusions Age, gender, Hb, Alb, Hcy and PU area were the risk factors of pressure ulcer healing.

Production of L-citrulline by a recombinant Corynebacterium crenatum SYPA 5-5 whole-cell biocatalyst / 生物工程学报

Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.

Whole-cell biotransformation for simultaneous synthesis of L-2-aminobutyric acid and D-gluconic acid in recombinant Escherichia coli / 生物工程学报

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, expressing L- threonine dehydratase from Escherichia coli, and co-expressing leucine dehydrogenase from Bacillus cereus and glucose dehydrogenase from Bacillus subtilis for cofactor regeneration, was constructed and used for one-pot production of L-2-aminobutyric acid (L-ABA) and D- gluconic acid from L-threonine and D-glucose. We used shake-flask culture to study the whole-cell catalytic condition including temperature, pH, proper permeabilization of cells and optimal wet cells amount. Moreover, the whole-cell catalyst was cultured in 5-L fermentor by fed-batch fermentation, and 164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid. The whole-cell catalyst is promising to fulfill industrial requirements for L-ABA and D-gluconic acid.

Analysis of real-time review and intervention on irrational outpatient infusion prescriptions / 中国基层医药

Objective To analyze type of irrational outpatient infusion prescriptions returned by clinical pharmacists'pre -prescription review and intervention in our hospital.Methods Outpatient infusion prescriptions reviewed by clinical pharmacists in 2014 were statistically analyzed in terms of type,constitution and rate of qualified prescriptions.Results Type of unstandard writing or input errors prescriptions accounted for 35.19%,which was the great majority of the irrational outpatient infusion prescriptions.The rate of qualified prescriptions was 99.15%,which was higher than 91.53% in 2011 when pre -prescription review and intervention had not implemented.Conclusion Pre -prescription review and intervention could improve the quality of outpatient infusion prescriptions.Finally,safety and effectiveness of outpatient infusion could be guaranteed.

Clinical Observation of Sodium Hyaluronate Combined with Glucosamine in the Treatment of Knee Osteoar-thritis / 中国药房

OBJECTIVE:To observe the clinical efficacy and safety of sodium hyaluronate combined with glucosamine in the treatment of knee osteoarthritis. METHODS:1 220 patients with knee osteoarthritis were randomly divided into observation group and control group. The patients in control group were treated with Sodium hyaluronate injection alone,once a week;while the ob-servation group was additionally and orally treated with Glucosamine hydrochloride capsules two tablets,three times a day. The course of treatment was 5 weeks in both groups. The relevant physicotherapeutics and oral medication in the treatment of the dis-ease should be stopped. The clinic efficacy,visual analog scale(VAS)and lysholm knee score before and after treatment,and inci-dence of adverse reaction were observed. RESULTS:Total effective rate of observation group was significantly higher than control group,with statistically significant difference(P<0.05). After treatment,VAS and lysholm knee scores in both groups were signifi-cantly lower than before,and observation group was lower than control group,with significant differences(P<0.05). There were no severe adverse reactions during treatment. CONCLUSIONS:Sodium hyaluronate combined with glucosamine has more signifi-cant clinical efficacy in the treatment of knee osteoarthritis than sodium hyaluronate alone,with good safety.

Effect of sarpogrelate hydrochloride on cytochrome P450 2D1/2 in rats / 中国药理学通报

Aim To investigate the influence of sarpog-relate hydrochloride (SH)on the pharmacokinetic pro-file of dextromethorphan (DM),the typical substrate of CYP2D1 /2,in rats when they were administered co-instantaneously.Methods A total of 1 2 SD rats were randomly divided into two groups:the control group (DM,1 0 mg·kg-1 )and the sarpogrelate group (SH, 1 0 mg·kg-1 ;DM,1 0 mg·kg-1 ),which received in-tragastric administration.Plasma samples were collected immediately before and at different time points after drug administration.A LC-MS /MS method was used to determine the concentrations of DM in rat plasma. Pharmacokinetic parameters were analyzed using Drug and Statistics (DAS 2.0).Results There were signif-icant differences in the pharmacokinetic parameters of DM,including T1 2 (2.49 h ±0.93 h vs 1 .47 h ±0.20 h,P <0.05 ),Cmax (325.7 μg·L -1 ±1 33.2 μg· L -1 vs 1 04.5μg·L -1 ±52.4 μg·L -1 ,P <0.05), AUC0 -t(785.5 μg·L -1 ·h ±451 .9 μg·L -1 ·h vs 244.8 μg·L -1 ·h ±1 68.3μg·L -1 ·h,P <0.05) and AUC0 -∞(804.7 μg·L -1 ·h ±445.6 μg·L -1 ·h vs 251 .4 μg·L -1 ·h ±1 73.4 μg·L -1 ·h,P<0.05 )between the two groups.Conclusion SH could significantly inhibit the elimination of DM,the substrate of CYP2D1 /2 in rats.

Effects of pH and oxygen supply on production of 2,3-butanediol from biodiesel-derived glycerol by Bacillus amyloliquefaciens / 生物工程学报

Bacillus amyloliquefaciens B10-127 was used to produce 2,3-butanediol (2,3-BD) from residual glycerol obtained from biodiesel synthesis. Important variables for 2,3-BD fermentation, pH and dissolved oxygen, were studied. When pH was maintained constant, the yield of 2,3-BD was inhibited. The highest 2,3-BD yields were achieved by fermentation without any pH control with an optimized initial pH 6.5. Batch fermentative production of 2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods by changing agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (micro), specific glucose consumption rate (q(s)) and specific 2,3-BD formation rate (q(p)), a three-stage agitation speed control strategy was proposed, aimed at achieving high concentration, high yield and high productivity of 2,3-BD. Maximum concentration of 2,3-BD reached 38.1 g/L, with the productivity of 1.06 g/(L x h), which were 14.8% and 63.1% over the best results from constant agitation speeds. In a pulse fed-batch fermentation, 2,3-BD concentration and productivity were significantly improved to 71.2 g/L and 0.99 g/(L x h), respectively. To our knowledge, these results were the highest for 2,3-BD production from biodiesel-derived glycerol.

Construction of a recombinant Escherichia coli BL21/ pET-28a-lpgad and the optimization of transformation conditions for the efficient production of gamma-aminobutyric acid / 生物工程学报

In order to enhance gamma-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results showed that the recombinant E. coli BL21 (pET-28a-lpgad) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. The optimum pH and temperature of the enzyme were pH 4.8 and 37 degrees C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated gamma-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of gamma-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of gamma-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the gamma-aminobutyric acid construction of the industrial applications.

Cloning, expression and characterization of N-acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation / 生物工程学报

N-Acetylornithine aminotransferase (EC 2.6.1.11, ACOAT) catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine, the forth step involved in the L-arginine biosynthetic pathways. We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization. ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5. Analysis of argD sequences revealed that only one ORF existed, which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa. The argD gene from C. crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C. crenatum SYPA. Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g. Subsequently, the expression plasmid pJCtac-CcargD was transformed into C. crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C. crenatum CCD. Further fermentative character of CCD1 was also analyzed. The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.

Efficacy of ginsenosides combined with prednisone in patients with systemic lupus erythematosus: a prospective, randomized, double-blind, placebo-controlled trial / 中西医结合学报

Background: The side effects of glucocorticoid in treatment of systemic lupus erythematosus (SLE) have been the focus of debate, and our preliminary study indicates that ginsenosides can enhance the efficacy of dexamethasone. Objective: To observe the effects of ginsenosides combined with prednisone in SLE patients. Design, setting, participants and interventions: A total of 60 SLE patients from Department of Rheumatology and Immunology, Changhai Hospital, Second Military Medical University, were randomly divided into treatment group and control group, with 30 patients in each group. Patients in the treatment group were given routine treatment with prednisone plus ginsenosides, while those in the control group were given routine treatment with prednisone plus placebo. They were all treated for 3 months. Main outcome measures: After three-month treatment, syndrome score in traditional Chinese medicine (TCM), total response rate and symptom improvement rate were measured and evaluated. Results: Twenty-eight cases in treatment group and twenty-seven cases in control group were included in analysis. The total response rates in the treatment group and control group were 89.28% and 66.67% respectively, and there was a significant difference between the two groups (P<0.05). After treatment, the TCM syndrome scores in the two groups were lower than those before treatment (P<0.01), and prednisone plus ginsenosides was better in decreasing the TCM syndrome score than prednisone plus placebo (P<0.05). The symptoms were improved in the treatment group as compared with the control group (P<0.05). Conclusion: Prednisone combined with ginsenosides can increase the clinical effective rate and improve the clinical symptoms of SLE patients.

The expression and meaning of liver X receptor in obstructive sleep apnea-hypopnea syndrome patients / 中国实用内科杂志

Objective To study the liver X receptor(LXR)and the gene expression of cholesterol ester transfer protein(CETP),cyclooxygenase-2(COX2)in obstructive sleep apnea hypopnea syndrome(OSAHS)patients.Methods Macrophages were separated from the blood drawn in 30 cases of OSAHS patients and 30 healthy control subjects in the morning,RT-PCR gel eletrophoresis was used to determine the patterns of gene expression.Results OSAHS patients had higher LXR,COX2,CETP expression(P

EFFECTS OF FU-TZE "801" ON SURVIVAL AND MICROCIRCU- LATORY CHANGES IN BURN SHOCK RATS / 中国药理学通报

The antishock properties of Fu-tze "801" , an active fraction isolated from the root of Aconite,were studied on burned rats. The rats were given 65-70% TB'SA second degree scalds. All animals died in a short time postburn. Using this rat model, it was found that the marked spasmodic rhythmic constriction in arterioles occurred and the microierculatory flow state showed pronounced derangements in the me-senteric microcirculation, and platelet aggregate increased in circulating blood.After treatment with Fu-tze "801" , the survival and the mirocirculation were improved in the scalded rats, these findings suggest that Fu-tze "801" affords the antishock action on rats after a major scald burn.