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1.
Chinese Journal of Biotechnology ; (12): 1564-1577, 2021.
Article in Chinese | WPRIM | ID: wpr-878655

ABSTRACT

As an important model industrial microorganism, Escherichia coli has been widely used in pharmaceutical, chemical industry and agriculture. In the past 30 years, a variety of new strategies and techniques, including artificial intelligence, gene editing, metabolic pathway assembly, and dynamic regulation have been used to design, construct, and optimize E. coli cell factories, which remarkably improved the efficiency for biotechnological production of chemicals. In this review, three key aspects for constructing E. coli cell factories, including pathway design, pathway assembly and regulation, and optimization of global cellular performance, are summarized. The technologies that have played important roles in metabolic engineering of E. coli, as well as their future applications, are discussed.


Subject(s)
Artificial Intelligence , Escherichia coli/genetics , Gene Editing , Metabolic Engineering , Metabolic Networks and Pathways/genetics
2.
Chinese Journal of Biotechnology ; (12): 1368-1375, 2021.
Article in Chinese | WPRIM | ID: wpr-878638

ABSTRACT

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.


Subject(s)
Bacterial Proteins , Diphtheria Toxin/genetics , Escherichia coli/genetics , Humans , Molecular Chaperones/genetics , Recombinant Proteins/genetics
3.
Chinese Journal of Biotechnology ; (12): 1334-1345, 2021.
Article in Chinese | WPRIM | ID: wpr-878635

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.


Subject(s)
COVID-19 , Codon/genetics , Cysteine Endopeptidases/genetics , Escherichia coli/genetics , Humans , Peptide Hydrolases , SARS-CoV-2 , Viral Nonstructural Proteins/genetics
4.
Chinese Journal of Biotechnology ; (12): 1287-1297, 2021.
Article in Chinese | WPRIM | ID: wpr-878631

ABSTRACT

L-Homoserine is a non-essential amino acid that is often used as an important platform compound and additive in industrial production. To improve the production efficiency, a previously constructed L-homoserine producing strain E. coli H0-0 was used as a chassis for further metabolic modification. Firstly, the ppc and pyccgP458S genes were overexpressed to optimize the Kreb's cycle. Subsequently, thrAC1034T and lysCcgC932T were overexpressed to improve the product synthesis, followed by inactivation of iclR gene to reduce the accumulation of by-products. The introduction of three sucrose metabolism genes, scrA, scrB and scrK, enabled E. coli to ferment sucrose. The titer of L-homoserine increased from 3.2 g/L to 11.1 g/L.


Subject(s)
Escherichia coli/genetics , Homoserine , Metabolic Engineering , Serine
5.
Chinese Journal of Biotechnology ; (12): 1277-1286, 2021.
Article in Chinese | WPRIM | ID: wpr-878630

ABSTRACT

Microbial cell factories capable of producing valuable chemicals from renewable feedstocks provide a promising alternative towards sustainability. However, environmental stress remarkably affects the performance of microbial cell factories. By extending the chronological lifespan of microbial cells, the performance of microbial cell factories can be greatly improved. Firstly, an evaluation system for chronological lifespan and semi-chronological lifespan was established based on the changes in survival rates. Secondly, the addition of anti-aging drugs such as cysteine, carnosine, aminoguanidine and glucosamine increased the chronological lifespan of E. coli by 80%, 80%, 50% and 120%, respectively. Finally, we demonstrated that extending the chronological lifespan of E. coli increased the yield of metabolites produced by E. coli cell factories with endogenous (lactic acid and pyruvic acid) or exogenous (malic acid) metabolic pathway by 30.0%, 25.0%, and 27.0%, respectively. The strategy of extending chronological lifespan of E. coli provides a potential approach for enhancing the performance of microbial cell factories.


Subject(s)
Escherichia coli/genetics , Lactic Acid , Longevity , Metabolic Engineering , Metabolic Networks and Pathways
6.
Chinese Journal of Biotechnology ; (12): 860-873, 2021.
Article in Chinese | WPRIM | ID: wpr-878601

ABSTRACT

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.


Subject(s)
Corynebacterium glutamicum/genetics , Escherichia coli/genetics , Metabolic Engineering , Metabolic Networks and Pathways/genetics
7.
Chinese Journal of Biotechnology ; (12): 593-603, 2021.
Article in Chinese | WPRIM | ID: wpr-878584

ABSTRACT

Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.


Subject(s)
Bread , Escherichia coli/genetics , Hydrogen Peroxide , Oxidoreductases , Triticum
8.
Chinese Journal of Biotechnology ; (12): 321-330, 2021.
Article in Chinese | WPRIM | ID: wpr-878565

ABSTRACT

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.


Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Genes, Reporter/genetics , Genetic Vectors/genetics , Lac Operon/genetics , Plasmids/genetics , beta-Galactosidase/genetics
9.
Chinese Journal of Biotechnology ; (12): 266-275, 2021.
Article in Chinese | WPRIM | ID: wpr-878560

ABSTRACT

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²⁺ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.


Subject(s)
Escherichia coli/genetics , Hexosyltransferases/genetics , Inulin , Oligosaccharides , Sucrose
10.
Chinese Journal of Biotechnology ; (12): 228-241, 2021.
Article in Chinese | WPRIM | ID: wpr-878557

ABSTRACT

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.


Subject(s)
Escherichia coli/genetics , Lactococcus , Metabolic Engineering , Pyrazines , Threonine
11.
Chinese Journal of Biotechnology ; (12): 196-206, 2021.
Article in Chinese | WPRIM | ID: wpr-878554

ABSTRACT

Polyhydroxyalkanoates (PHAs) have obtained much attention in biomaterial fields due to their similar physicochemical properties to those of the petroleum-derived plastics. Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is one member of the PHAs family, and has better toughness and transparency compared to existing polylactic acid (PLA) and poly[(R)-3-hydroxybutyrate] [P(3HB)]. First, we confirmed the one-step biosynthesis of P(LA-co-3HB) with the lactate fraction of 23.8 mol% by introducing P(3HB-co-LA) production module into Escherichia coli MG1655. Then, the lactate fraction was increased to 37.2 mol% in the dld deficient strain WXJ01-03. The genes encoding the thioesterases, ydiI and yciA, were further knocked out, and the lactate fraction in the P(3HB-co-LA) was improved to 42.3 mol% and 41.1 mol% respectively. Strain WXJ03-03 with dld, ydiI and yciA deficient was used for the production of the LA-enriched polymer, and the lactate fraction was improved to 46.1 mol%. Notably, the lactate fraction in P(3HB-co-LA) from xylose was remarkably higher than from glucose, indicating xylose as a potent carbon source for P(3HB-co-LA) production. Therefore, the deficiency of thioesterase may be considered as an effective strategy to improve the lactate fraction in P(3HB-co-LA) in xylose fermentation.


Subject(s)
Escherichia coli/genetics , Hydroxybutyrates , Lactic Acid , Polyesters , Polyhydroxyalkanoates , Xylose
12.
Electron. j. biotechnol ; 46: 8-13, jul. 2020. tab, graf
Article in English | LILACS | ID: biblio-1223212

ABSTRACT

BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.


Subject(s)
Escherichia coli/metabolism , Hydroxybutyrates/metabolism , Repressor Proteins/genetics , Biopolymers/genetics , Recombinant Proteins , RNA-Binding Proteins/genetics , Gene Deletion , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Metabolic Engineering , Ligases/metabolism
13.
Chinese Journal of Biotechnology ; (12): 2868-2876, 2020.
Article in Chinese | WPRIM | ID: wpr-878536

ABSTRACT

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.


Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Humans , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Tissue Inhibitor of Metalloproteinase-2/genetics
14.
Chinese Journal of Biotechnology ; (12): 2467-2477, 2020.
Article in Chinese | WPRIM | ID: wpr-878503

ABSTRACT

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.


Subject(s)
Anabaena/genetics , Cloning, Molecular , DNA-Directed RNA Polymerases , Escherichia coli/genetics , Gene Expression , Mercury , Plasmids , Viral Proteins
15.
Chinese Journal of Biotechnology ; (12): 2367-2376, 2020.
Article in Chinese | WPRIM | ID: wpr-878493

ABSTRACT

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.


Subject(s)
Ammonia-Lyases/genetics , Escherichia coli/genetics , Propionates , Rhodotorula , Tyrosine/genetics
16.
Chinese Journal of Biotechnology ; (12): 2104-2112, 2020.
Article in Chinese | WPRIM | ID: wpr-878470

ABSTRACT

Shikimic acid is an intermediate metabolite in the synthesis of aromatic amino acids in Escherichia coli and a synthetic precursor of Tamiflu. The biosynthesis of shikimic acid requires blocking the downstream shikimic acid consuming pathway that leads to inefficient production and cell growth inhibition. In this study, a dynamic molecular switch was constructed by using growth phase-dependent promoters and degrons. This dynamic molecular switch was used to uncouple cell growth from shikimic acid synthesis, resulting in the production of 14.33 g/L shikimic acid after 72 h fermentation. These results show that the dynamic molecular switch could redirect the carbon flux by regulating the abundance of target enzymes, for better production.


Subject(s)
Escherichia coli/genetics , Escherichia coli Proteins/genetics , Industrial Microbiology/methods , Metabolic Engineering , Shikimic Acid/metabolism
17.
Chinese Journal of Biotechnology ; (12): 2066-2075, 2020.
Article in Chinese | WPRIM | ID: wpr-878466

ABSTRACT

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.


Subject(s)
Animals , Antigens, Viral/genetics , Biological Assay , Chickens/immunology , Escherichia coli/genetics , Infectious bursal disease virus/immunology , Poultry Diseases , Vaccines, Synthetic/isolation & purification , Viral Structural Proteins/immunology , Viral Vaccines/immunology
18.
Mem. Inst. Oswaldo Cruz ; 115: e190347, 2020. tab, graf
Article in English | LILACS, SES-SP | ID: biblio-1135231

ABSTRACT

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.


Subject(s)
Animals , Female , Mice , BCG Vaccine/immunology , Mycobacterium smegmatis/immunology , Green Fluorescent Proteins/immunology , Escherichia coli/immunology , Genetic Vectors/immunology , Mycobacterium bovis/immunology , Escherichia coli/genetics , Genetic Vectors/genetics , Mice, Inbred BALB C
19.
Rev. Soc. Bras. Med. Trop ; 53: e20190429, 2020. tab, graf
Article in English | LILACS | ID: biblio-1057306

ABSTRACT

Abstract INTRODUCTION: Nosocomial and community acquired urinary tract infections (UTIs) are one of the most encountered infections in the world. METHODS: This study aimed to determine the antibiotic susceptibility, phylogeny, and virulence genes of 153 Escherichia coli strains isolated from UTIs. Antimicrobial susceptibility of the isolates to different classes of antimicrobials was determined by the VITEK-2 automated system. Presence of virulence genes and phylogenetic groups were investigated by PCR. RESULTS: Regarding susceptibility to antimicrobials, ampicillin resistance was most abundant (67.3%), followed by amoxicillin-clavulanic acid (50.9%); least abundant was resistance to amikacin (1.3%) and nitrofurantoin (1.3%). Multi drug resistance (MDR) was observed in 34.6% of the isolates, and all isolates were found to be susceptible to imipenem, meropenem and fosfomycine. The majority of the isolates belonged to the phylogenetic group B23 (35.9%), followed by A1 (20.9%), D1 (18.9%), D2 (12.4%), A0 (%5.9), B1 (3.9%) and B2 (1.9%). Among E. coli strains examined, 49% had iucD, 32.7% papE-F, 26.1% papC, 15% cnf2, 11.1% sfa, 7.8% cnf1, 1.3% afaE, 1.3% afaD, 1.3% hlyA, 0.7% f17a-A, 0.7% clpG and 0.7% eaeA genes. CONCLUSIONS Our research demonstrated that virulence factors were distributed among different phylogroup/subgroups, which play a role in UTIs pathogenesis in humans. For this reason, complex and detailed studies are required to determine the relationship between virulence factors and specific E. coli strains that cause UTIs in humans.


Subject(s)
Humans , Urinary Tract Infections/microbiology , Virulence Factors/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Infections/microbiology , Anti-Bacterial Agents/pharmacology , Phylogeny , RNA, Ribosomal, 16S , Microbial Sensitivity Tests , Polymerase Chain Reaction , Escherichia coli/isolation & purification , Genotype
20.
Rev. Inst. Nac. Hig ; 50(1-2): 4-13, Diciembre 2019. Tab
Article in Spanish | LILACS, LIVECS | ID: biblio-1118292

ABSTRACT

Las infecciones del tracto urinario afectan al ser humano a lo largo de su vida y son frecuentes tanto en el ámbito comunitario como en el nosocomial. El objetivo de este estudio fue Identificar los principales agentes etiológicos y el perfil de resistencia a los antibióticos, presentado por los microorganismos más frecuentemente aislados de los urocultivos de pacientes con infección urinaria que acudieron al Laboratorio "Luis Razetti" Mérida -Venezuela, entre enero y junio de 2015. Este estudio fue de tipo observacional, de corte transversal y descriptivo. La población y muestra estuvo conformada por 149 pacientes de ambos sexos, cuyas muestras de orina fueron procesadas utilizado el método del asa calibrada y la identificación bacteriana mediante pruebas bioquímicas convencionales. La susceptibilidad antimicrobiana se determinó a través del método de difusión del disco en agar. Escherichia coli predominó en un 84,6 %, seguido de Proteus mirabilis y Enterococcus faecalis, ambos con (4,7 %). Los porcentajes más altos de resistencia para los aislados de E. coli, se observaron para ampicilina (92,06 %), ampicilina/sulbactam (68,25 %), ácido nalidíxico (38,89 %), ciprofloxacina (38,89 %) y trimetroprim­sulfametoxazol (54,76 %); y presentaron altos niveles de sensibilidad a Nitrofurantoína (80,95 %). El 5,15 % de las cepas de E. coli se mostraron fenotípicamente productoras de belalactamasa de espectro extendido y el 35,29 % de las otras Enterobacteriaceae aisladas, presentaron un perfil fenotípico compatible con la producción de la enzima Inhibitory-resistant TEM (IRT). Es importante destacar que estos estudios permiten conocer la etiología a de infecciones urinarias en la comunidad, así como los perfiles de resistencia y sensibilidad a nivel local, datos relevantes para establecer pautas de tratamiento empírico adaptadas a cada medio.


Urinary tract infections affect the human being throughout his life and are among the most frequent in both the community and nosocomial settings. The Aim of this study was to Identify the main etiological agents and antibiotic resistance profile presented by isolated microorganisms in the urocultures of patients with urinary tract infection who attended the Laboratory "Luis Razetti" Mérida -Venezuela, between January and June 2015. This study was observational, cross-sectional and descriptive. The population and sample consisted of 149 patients of both sexes, whose urine samples were processed using the calibrated handle method and bacterial identification through conventional biochemical tests. The antimicrobial susceptibility is determined through the disk diffusion method in agar. Escherichia coli dominated by 84.6 %, followed by Proteus mirabilis and Enterococcus faecalis, both with (4.7 %). The highest percentages of resistance for E. coli were observed for ampicillin (92.06 %), ampicillin/sulbactam (68.25 %), nalidixic acid (38.89 %), ciprofloxacin (38.89 %) trimetroprim-sulfamethoxazole (54.76 %); and had high levels of sensitivity to Nitrofurantoin (80.95 %). 5.15 % of E. coli strains were phenotypically producing extended-spectrum belalactamase and 35.29 % of others Enterobacteriaceae isolated had a phenotypic profile compatible with the production of the Enzyme Inhibitoryresistant TEM (IRT). It is important to note that these studies allow knowing the etiology of urinary tract infections in the community as well as resistance and sensitivity profiles at the local level, relevant data to establish empirical processing guidelines tailored to each medium.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Young Adult , Urinary Tract Infections/microbiology , Urinary Tract Infections/drug therapy , Drug Resistance, Bacterial , Escherichia coli/isolation & purification , Anti-Bacterial Agents/therapeutic use , Phenotype , Cross-Sectional Studies , Community-Acquired Infections/microbiology , Community-Acquired Infections/drug therapy , Escherichia coli/drug effects , Escherichia coli/genetics , Anti-Bacterial Agents/pharmacology
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