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1.
Weiyu Wang Jiaqi Sun Wenjun Xiao Li Jiang Ruyue Wang Jun Fan 《Biotechnology letters》2017,39(11):1733-1740
Objectives
To optimize the production of active inclusion bodies (IBs) containing human d-amino acid oxidase (hDAAO) in Escherichia coli.Results
The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing d-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing d-alanine, d-serine and d-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the l-serine racemization activity of the bacterially expressed maize serine racemase.Conclusions
Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.2.
Hongchao Wang Chen Zhang Haiqin Chen Qin Yang Xin Zhou Zhennan Gu Hao Zhang Wei Chen Yong Q. Chen 《Biotechnology letters》2016,38(10):1761-1768
3.
Qibo Zhang Lisa A. Ford Anne M. Evans Douglas R. Toal 《Metabolomics : Official journal of the Metabolomic Society》2017,13(8):92
Introduction
A major bottleneck in metabolomic studies is metabolite identification from accurate mass spectrometric data. Metabolite x17299 was identified in plasma as an unknown in a metabolomic study using a compound-centric approach where the associated ion features of the compound were used to determine the true molecular mass.Objectives
The aim of this work is to elucidate the chemical structure of x17299, a new compound by de novo interpretation of mass spectrometric data.Methods
An Orbitrap Elite mass spectrometer was used for acquisition of mass spectra up to MS4 at high resolution. Synthetic standards of N,N,N-trimethyl-l-alanyl-l-proline betaine (l,l-TMAP), a diastereomer, and an enantiomer were chemically prepared.Results
The planar structure of x17299 was successfully proposed by de novo mechanistic interpretation of mass spectrometric data without any laborious purification and nuclear magnetic resonance spectroscopic analysis. The proposed structure was verified by deuterium exchanged mass spectrometric analysis and confirmed by comparison to a synthetic standard. Relative configuration of x17299 was determined by direct chromatographic comparison to a pair of synthetic diastereomers. Absolute configuration was assigned after derivatization of x17299 with a chiral auxiliary group followed by its chromatographic comparison to a pair of synthetic standards.Conclusion
The chemical structure of metabolite x17299 was determined to be l,l-TMAP.4.
Objective
Develop and validate particular, concrete, and abstract yet plausible in silico mechanistic explanations for large intra- and interindividual variability observed for eleven bioequivalence study participants. Do so in the face of considerable uncertainty about mechanisms.Methods
We constructed an object-oriented, discrete event model called subject (we use small caps to distinguish computational objects from their biological counterparts). It maps abstractly to a dissolution test system and study subject to whom product was administered orally. A subject comprises four interconnected grid spaces and event mechanisms that map to different physiological features and processes. Drugs move within and between spaces. We followed an established, Iterative Refinement Protocol. Individualized mechanisms were made sufficiently complicated to achieve prespecified Similarity Criteria, but no more so. Within subjects, the dissolution space is linked to both a product-subject Interaction Space and the GI tract. The GI tract and Interaction Space connect to plasma, from which drug is eliminated.Results
We discovered parameterizations that enabled the eleven subject simulation results to achieve the most stringent Similarity Criteria. Simulated profiles closely resembled those with normal, odd, and double peaks. We observed important subject-by-formulation interactions within subjects.Conclusion
We hypothesize that there were interactions within bioequivalence study participants corresponding to the subject-by-formulation interactions within subjects. Further progress requires methods to transition currently abstract subject mechanisms iteratively and parsimoniously to be more physiologically realistic. As that objective is achieved, the approach presented is expected to become beneficial to drug development (e.g., controlled release) and to a reduction in the number of subjects needed per study plus faster regulatory review.5.
Hui-Min Qin Songtao Li Yu-Fu Zhang Jian-Wen Wang Jixuan Li Shiyi Song Fuping Lu Yu Li 《Biotechnology letters》2016,38(10):1747-1752
Objectives
To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from d-mannose by two-step fermentation pathway in Escherichia coli.Results
E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using d-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation.Conclusions
Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.6.
Wei-Wei Li Yan Yang Qi-Gang Dai Li-Li Lin Tong Xie Li-Li He Jia-Lei Tao Jin-Jun Shan Shou-Chuan Wang 《Metabolomics : Official journal of the Metabolomic Society》2018,14(7):90
Introduction
Neonatal cholestatic disorders are a group of hepatobiliary diseases occurring in the first 3 months of life. The most common causes of neonatal cholestasis are infantile hepatitis syndrome (IHS) and biliary atresia (BA). The clinical manifestations of the two diseases are too similar to distinguish them. However, early detection is very important in improving the clinical outcome of BA. Currently, a liver biopsy is the only proven and effective method used to differentially diagnose these two similar diseases in the clinic. However, this method is invasive. Therefore, sensitive and non-invasive biomarkers are needed to effectively differentiate between BA and IHS. We hypothesized that urinary metabolomics can produce unique metabolite profiles for BA and IHS.Objectives
The aim of this study was to characterize urinary metabolomic profiles in infants with BA and IHS, and to identify differences among infants with BA, IHS, and normal controls (NC).Methods
Urine samples along with patient characteristics were obtained from 25 BA, 38 IHS, and 38 NC infants. A non-targeted gas chromatography–mass spectrometry (GC–MS) metabolomics method was used in conjunction with orthogonal partial least squares discriminant analysis (OPLS-DA) to explore the metabolomic profiles of BA, IHS, and NC infants.Results
In total, 41 differentially expressed metabolites between BA vs. NC, IHS vs. NC, and BA vs. IHS were identified. N-acetyl-d-mannosamine and alpha-aminoadipic acid were found to be highly accurate at distinguishing between BA and IHS.Conclusions
BA and IHS infants have specific urinary metabolomic profiles. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be used to discriminate BA from IHS.7.
Weirui Zhao Huanru Ding Sheng Hu Jun Huang Changjiang Lv Jiaqi Mei Zhihua Jin Shanjing Yao Lehe Mei 《Biotechnology letters》2018,40(7):1049-1055
Objective
To develop a new and efficient biocatalytic synthesis method of imidazole-4-acetic acid (IAA) from l-histidine (l-His).Results
l-His was converted to imidazole-4-pyruvic acid (IPA) by an Escherichia coli whole-cell biocatalyst expressing membrane-bound l-amino acid deaminase (ml-AAD) from Proteus vulgaris firstly. The obtained IPA was subsequently decarboxylated to IAA under the action of H2O2. Under optimum conditions, 34.97 mM IAA can be produced from 50 mM l-His, with a yield of 69.9%.Conclusions
Compared to the traditional chemical synthesis, this biocatalytic method for IAA production is not only environmentally friendly, but also more cost effective, thus being promising for industrial IAA production.8.
Pan Cui Tong-Yi Dou Shi-Yang Li Jun-Xia Lu Li-Wei Zou Ping Wang Yan-Ping Sun Da-Cheng Hao Guang-Bo Ge 《Biotechnology letters》2016,38(8):1367-1373
Objectives
To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin.Results
Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-l-rhamnosidase activity while the β-d-glucosidase activity was abrogated. The addition of Mg2+, Fe2+ and Co2+ was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and 1H-NMR.Conclusion
A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-l-rhamnosidase activity of naringinase and blocked its β-d-glucosidase activity.9.
Background
Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.Results
Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.Conclusion
An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.10.
Gesiane Tavares Gabriela Venturini Kallyandra Padilha Roberto Zatz Alexandre C. Pereira Ravi I. Thadhani Eugene P. Rhee Silvia M. O. Titan 《Metabolomics : Official journal of the Metabolomic Society》2018,14(4):39
Introduction
Metabolomics allows exploration of novel biomarkers and provides insights on metabolic pathways associated with disease. To date, metabolomics studies on CKD have been largely limited to Caucasian populations and have mostly examined surrogate end points.Objective
In this study, we evaluated the role of metabolites in predicting a primary outcome defined as dialysis need, doubling of serum creatinine or death in Brazilian macroalbuminuric DKD patients.Methods
Non-targeted metabolomics was performed on plasma from 56 DKD patients. Technical triplicates were done. Metabolites were identified using Agilent Fiehn GC/MS Metabolomics and NIST libraries (Agilent MassHunter Work-station Quantitative Analysis, version B.06.00). After data cleaning, 186 metabolites were left for analyses.Results
During a median follow-up time of 2.5 years, the PO occurred in 17 patients (30.3%). In non-parametric testing, 13 metabolites were associated with the PO. In univariate Cox regression, only 1,5-anhydroglucitol (HR 0.10; 95% CI 0.01–0.63, p?=?.01), norvaline and l-aspartic acid were associated with the PO. After adjustment for baseline renal function, 1,5-anhydroglucitol (HR 0.10; 95% CI 0.02–0.63, p?=?.01), norvaline (HR 0.01; 95% CI 0.001–0.4, p?=?.01) and aspartic acid (HR 0.12; 95% CI 0.02–0.64, p?=?.01) remained significantly and inversely associated with the PO.Conclusion
Our results show that lower levels of 1,5-anhydroglucitol, norvaline and l-aspartic acid are associated with progression of macroalbuminuric DKD. While norvaline and l-aspartic acid point to interesting metabolic pathways, 1,5-anhydroglucitol is of particular interest since it has been previously shown to be associated with incident CKD. This inverse biomarker of hyperglycemia should be further explored as a new tool in DKD.11.
D. Jacob C. Deborde M. Lefebvre M. Maucourt A. Moing 《Metabolomics : Official journal of the Metabolomic Society》2017,13(4):36
Introduction
Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.Objectives
The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.Methods
NMRProcFlow was developed to be a graphical and interactive 1D NMR (1H & 13C) spectra processing tool.Results
NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.Conclusion
Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.12.
Jinjin Xu Yajun Bai Taiping Fan Xiaohui Zheng Yujie Cai 《Biotechnology letters》2017,39(10):1559-1566
Objectives
To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).Results
The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with d -alanine the most preferred substrate. The K m and V max values for d -alanine were 9 mM and 20 μmol min?1 mg?1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.Conclusions
d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.13.
Ying Wang Guo-Si Li Pei Qiao Ling Lin Hai-Long Xue Li Zhu Mian-Bin Wu Jian-Ping Lin Li-Rong Yang 《Biotechnology letters》2018,40(11-12):1551-1559
Objective
To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).Results
l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.Conclusions
By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.14.
Justin J. J. van der Hooft Wejdan Alghefari Eleanor Watson Paul Everest Fraser R. Morton Karl E. V. Burgess David G. E. Smith 《Metabolomics : Official journal of the Metabolomic Society》2018,14(11):144
Introduction
Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity.Objectives
For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium.Methods
To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h.Results
This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy.Conclusions
Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.15.
Lia Bally Cédric Bovet Christos T. Nakas Thomas Zueger Jean-Christophe Prost Jean-Marc Nuoffer Alexander B. Leichtle Georg Martin Fiedler Christoph Stettler 《Metabolomics : Official journal of the Metabolomic Society》2017,13(7):78
Introduction
Exercise-associated metabolism in type 1 diabetes (T1D) remains under-studied due to the complex interplay between exogenous insulin, counter-regulatory hormones and insulin-sensitivity.Objective
To identify the metabolic differences induced by two exercise modalities in T1D using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HRMS) based metabolomics.Methods
Twelve T1D adults performed intermittent high-intensity (IHE) and continuous-moderate-intensity (CONT) exercise. Serum samples were analysed by UHPLC–HRMS.Results
Metabolic profiling of IHE and CONT highlighted exercise-induced changes in purine and acylcarnitine metabolism.Conclusion
IHE may increase beta-oxidation through higher ATP-turnover. UHPLC–HRMS based metabolomics as a data-driven approach without an a priori hypothesis may help uncover distinctive metabolic effects during exercise in T1D.Clinical trial registration number is www.clinicaltrials.gov: NCT02068638.16.
Mu Wang Qiande Liang Han Li Wei Xia Jie Li Yang Peng Yuanyuan Li Zengchun Ma Bing Xu Yue Gao Shunqing Xu 《Metabolomics : Official journal of the Metabolomic Society》2016,12(8):131
Introduction
The maternal body often faces unique physiological challenges in amino acid metabolism due to the continuous requirement of nutrients and substrates for fetal development and additional energy stores for labor and lactation during pregnancy.Objective
The aims of the present study is to find out the metabolites involved in amino acid metabolism in a large longitudinal healthy pregnant cohort and provide baseline data for future studies of pregnancy and disease from in utero environmental stress factors.Method
We conducted a UPLC-QTOFMS based-urine metabolomics study to investigate the dynamic amino acid metabolic profiles and pathways of 232 healthy pregnant women in their first, second and third trimesters. After multivariate classification to select the metabolites with the strongest contributions to dynamic alterations in normal pregnancy, we applied the method of standard deviation step (SDSD) down for statistical significance analysis to enhance the value of metabolites in clinical practice.Results
Kynurenic acid, an endogenous antagonist of N-methyl-d-aspartate receptors, increased significantly in middle pregnancy. l-aspartyl-4-phosphate, a potential marker for lower tolerance against fatigue of human body, decreased significantly in the third trimester. Cysteinylglycine, a pyrolysis product of glutathione, significantly increased in late pregnancy. These findings presented a novel insight into normal pregnancy-related regulation of the generation of excitatory neurotransmitter receptor antagonists, maternal fatigue, oxidative stress and so on.Conclusion
This normal pregnancy related amino acid metabolic profile as well as the pathways information might be valuable to explore the complex mechanisms of physiological metabolic challenge in amino acid metabolism with the potential capacity to generate a novel hypothesis, which in turn could provide an ideal start for a large-scale epidemiological study of women who subsequently develop diseases, e.g., gestational depression.17.
Rabbit plasma metabolomic analysis of Nitroproston®: a multi target natural prostaglandin based-drug
Ksenia Shestakova Alex Brito Natalia V. Mesonzhnik Natalia E. Moskaleva Ksenia O. Kurynina Natalia M. Grestskaya Igor V. Serkov Igor I. Lyubimov Vladimir V. Bezuglov Svetlana A. Appolonova 《Metabolomics : Official journal of the Metabolomic Society》2018,14(9):112
Introduction
Nitroproston® is a novel multi-target drug bearing natural prostaglandin E2 (PGE2) and nitric oxide (NO)-donating fragments for treatment of inflammatory and obstructive diseases (i.e., asthma and obstructive bronchitis).Objectives
To investigate the effects of Nitroproston® administration on plasma metabolomics in vivo.Methods
Experimental in vivo study randomly assigning the target drug (treatment group) or a saline solution without the drug (vehicle control group) to 12 rabbits (n?=?6 in each group). Untargeted (5880 initial features; 1869 negative–4011 positive ion peaks; UPLC–IT–TOF/MS) and 84 targeted moieties (Nitroproston® related metabolites, prostaglandins, steroids, purines, pyrimidines and amino acids; HPLC–QQQ–MS/MS) were measured from plasma at 0, 2, 4, 6, 8, 12, 18, 24, 32 and 60 min after administration.Results
PGE2, 13,14-dihydro-15-keto-PGE2, PGB2, 1,3-GDN and 15-keto-PGE2 increased in the treatment group. Steroids (i.e., cortisone, progesterone), organic acids, 3-oxododecanoic acid, nicotinate d-ribonucleoside, thymidine, the amino acids serine and aspartate, and derivatives pyridinoline, aminoadipic acid and uric acid increased (p?<?0.05 AUCROC curve?>?0.75) after treatment. Purines (i.e., xanthine, guanine, guanosine), bile acids, acylcarnitines and the amino acids l-tryptophan and l-phenylalanine were decreased. Nitroproston® impacted steroidogenesis, purine metabolism and ammonia recycling pathways, among others.Conclusion
Nitroproston®, a multi action novel drug based on natural prostaglandins, altered metabolites (i.e., guanine, adenine, cortisol, cortisone and aspartate) involved in purine metabolism, urea and ammonia biological cycles, steroidogenesis, among other pathways. Suggested mechanisms of action, metabolic pathway interconnections and useful information to further understand the metabolic effects of prostaglandin administration are presented.18.
19.
Objective
To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.Results
Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-d-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-β-d-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant.Conclusions
The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.20.