scholarly journals High-Yield Resveratrol Production in Engineered Escherichia coli

2011 ◽  
Vol 77 (10) ◽  
pp. 3451-3460 ◽  
Author(s):  
Chin Giaw Lim ◽  
Zachary L. Fowler ◽  
Thomas Hueller ◽  
Steffen Schaffer ◽  
Mattheos A. G. Koffas
Keyword(s):  
Escherichia Coli ◽  
High Yield ◽  
Plant Polyphenols ◽  
Content Type ◽  
Scientific Investigations ◽  
Highly Active ◽  
Structure Activity ◽  
Health Promoting ◽  
The Subject ◽  
Further Development

ABSTRACTPlant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create anEscherichia colistrain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.

scholarly journals Antimalarial Activity of the Myxobacterial Macrolide Chlorotonil A

2014 ◽  
Vol 58 (11) ◽  
pp. 6378-6384 ◽  
Author(s):  
Jana Held ◽  
Tamirat Gebru ◽  
Markus Kalesse ◽  
Rolf Jansen ◽  
Klaus Gerth ◽  
...  
Keyword(s):  
Screening Program ◽  
Antimalarial Activity ◽  
Stage Iv ◽  
Short Exposure ◽  
Parasite Development ◽  
Content Type ◽  
Highly Active ◽  
New Antibiotics ◽  
Further Development ◽  
Antimalarial Action

ABSTRACTMyxobacteria are Gram-negative soil-dwelling bacteria belonging to the phylumProteobacteria. They are a rich source of promising compounds for clinical application, such as epothilones for cancer therapy and several new antibiotics. In the course of a bioactivity screening program of secondary metabolites produced bySorangium cellulosumstrains, the macrolide chlorotonil A was found to exhibit promising antimalarial activity. Subsequently, we evaluated chlorotonil A againstPlasmodium falciparumlaboratory strains and clinical isolates from Gabon. Chlorotonil A was highly active, with a 50% inhibitory concentration between 4 and 32 nM; additionally, no correlations between the activities of chlorotonil A and artesunate (rho, 0.208) or chloroquine (rho, −0.046) were observed.Per ostreatment ofPlasmodium berghei-infected mice with four doses of as little as 36 mg of chlorotonil A per kg of body weight led to the suppression of parasitemia with no obvious signs of toxicity. Chlorotonil A acts against all stages of intraerythrocytic parasite development, including ring-stage parasites and stage IV to V gametocytes, and it requires only a very short exposure to the parasite to exert its antimalarial action. Conclusively, chlorotonil A has an exceptional and unprecedented profile of action and represents an urgently required novel antimalarial chemical scaffold. Therefore, we propose it as a lead structure for further development as an antimalarial chemotherapeutic.

scholarly journals Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Daisuke Koma ◽  
Takahiro Kishida ◽  
Eisuke Yoshida ◽  
Hiroyuki Ohashi ◽  
Hayato Yamanaka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aromatic Compound ◽  
Aromatic Compounds ◽  
High Yield ◽  
Rational Approach ◽  
Chromosome Engineering ◽  
Phenyllactic Acid ◽  
Content Type ◽  
E Coli ◽  
Free Strains

ABSTRACT Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr, aroL, and pheAfbr), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript “fbr” indicates that the enzyme encoded by the gene was feedback resistant. The generated strain could be changed to be a superior tyrosine-producing strain by replacing pheAfbr with tyrAfbr. A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr, and pheAfbr) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields. IMPORTANCE Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the E. coli chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).

scholarly journals Analysis of Serial Isolates of mcr-1-Positive Escherichia coli Reveals a Highly Active ISApl1 Transposon

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Erik Snesrud ◽  
Ana C. Ong ◽  
Brendan Corey ◽  
Yoon I. Kwak ◽  
Robert Clifford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Single Copy ◽  
The United States ◽  
Content Type ◽  
E Coli ◽  
Highly Active ◽  
Copy Numbers ◽  
Genes Encoding

ABSTRACT The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1. Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Virulent Bacteriophages Can Target O104:H4 Enteroaggregative Escherichia coli in the Mouse Intestine

2012 ◽  
Vol 56 (12) ◽  
pp. 6235-6242 ◽  
Author(s):  
Damien Maura ◽  
Matthieu Galtier ◽  
Chantal Le Bouguénec ◽  
Laurent Debarbieux
Keyword(s):  
Escherichia Coli ◽  
Dependent Manner ◽  
Content Type ◽  
Intestinal Pathogens ◽  
Colonization Model ◽  
Mouse Intestine ◽  
Further Development ◽  
Dose Dependent

ABSTRACTIn vivobacteriophage targeting of enteroaggregativeEscherichia coli(EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophagesin vivofor the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers.

scholarly journals Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

2015 ◽  
Vol 81 (13) ◽  
pp. 4458-4476 ◽  
Author(s):  
Christian Bille Jendresen ◽  
Steen Gustav Stahlhut ◽  
Mingji Li ◽  
Paula Gaspar ◽  
Solvej Siedler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Cinnamic Acid ◽  
Aromatic Compounds ◽  
Physcomitrella Patens ◽  
Coumaric Acid ◽  
Content Type ◽  
Highly Active ◽  
Enhanced Production ◽  
Wide Range

ABSTRACTPhenylalanine and tyrosine ammonia-lyases form cinnamic acid andp-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequencesin silicousing synteny information and aiming for sequence divergence. We performed a comparativein vivostudy, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production ofp-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacteriumBrevibacillus laterosporus, a bacterial-type enzyme from the amoebaDictyostelium discoideum, and a phenylalanine ammonia-lyase from the mossPhyscomitrella patens(producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium usingEscherichia colias the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes fromHerpetosiphon aurantiacusandFlavobacterium johnsoniaeresulted in high production ofp-coumaric acid inEscherichia coli(producing 440 μMp-coumaric acid OD600unit−1in the medium) and inLactococcus lactis. The enzymes were also efficient inSaccharomyces cerevisiae, wherep-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

scholarly journals Complete Genome Sequence of Escherichia coli BL21-AI

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Niketa Bhawsinghka ◽  
Katie F. Glenn ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Expression System ◽  
High Yield ◽  
Content Type ◽  
System A ◽  
Phage T7 ◽  
High Level

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

Problems of administrative law in the system of public administration

2018 ◽  
Vol 60 (6) ◽  
pp. 1255-1271
Author(s):  
Djamilya Ospanova ◽  
Duman Kussainov ◽  
Akif Suleimanov ◽  
Ainur Kussainov ◽  
Rysgul Abilsheyeva
Keyword(s):  
Local Government ◽  
Public Administration ◽  
Administrative Law ◽  
Content Type ◽  
Governmental Organization ◽  
State Authority ◽  
Regulatory Impact ◽  
The Subject ◽  
The Relationship ◽  
Further Development

Purpose The relevance of the work is determined by the fact that administrative law appears as one of the significant factors influencing the development of both the public administration system as a whole and the processes of its reform that the Russian Federation has faced today. The authors show that if the relations of state administration are the subject of the administrative law’s influence (after all, it is they who are influenced by administrative and legal norms), then we are faced with an extremely important question regarding the character and specificity of the relationship between administrative and legal relations and relations of state management, on the one hand, and analysis of the concept of administrative and legal regulation of public administration relations on the other. This goal is important, given that the authors substantiate the relationship between administrative law and public administration through identifying the possibilities of regulatory influence on the part of administrative law on public administration relations. Design/methodology/approach Prospects for the further development of the study are the formation of an understanding that government is not limited to administrative law, but it cannot be denied that the regulatory impact of law in public administration is decisive, and therefore public administration is largely a state-legal category, although it is not limited to this. Findings So, having analyzed the above approaches and positions of scientists regarding the subject of administrative law, the authors believe that it can include any managerial activity of the state authority and local government bodies that does not directly concern the subject matter of another branch of law; is realized with the help of the executive-administrative mechanism (in this case the author does not consider the executive-administrative mechanism as an exclusive prerogative or a unique feature of the executive authorities, although, of course, he agrees that it is the most typical and characteristic feature for them); is realized within the framework of a certain state authority, local government or non-governmental organization (in case of delegating state powers to it) to ensure its proper functioning (internal management relations); is implemented outside the framework of a certain state authority, local government or non-governmental organization (in case of delegating state powers to it) and is directed to other (external) with respect to the relevant body or organization of entities (external organizational management relations); and is largely characterized by relative constancy and immediacy of implementation relative to the management object. Originality/value Prospects for the further development of the study are the formation of an understanding that government is not limited to administrative law, but it cannot be denied that the regulatory impact of law in public administration is decisive, and therefore public administration is largely a state-legal category, although it is not limited to this. At the same time, indistinctness and uncertainty about different ways of understanding public administration often generate negative effects both at the general scientific level and at a purely practical level (when it comes to the exercise of administrative powers by certain state authorities).

scholarly journals Dihydroquinazolinone Inhibitors of Proliferation of Blood and Liver Stage Malaria Parasites

2013 ◽  
Vol 58 (3) ◽  
pp. 1516-1522 ◽  
Author(s):  
Emily R. Derbyshire ◽  
Jaeki Min ◽  
W. Armand Guiguemde ◽  
Julie A. Clark ◽  
Michele C. Connelly ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Plasmodium Berghei ◽  
Mammalian Cells ◽  
Blood Stage ◽  
Malaria Parasites ◽  
Liver Stage ◽  
Content Type ◽  
Minimal Toxicity ◽  
Structure Activity ◽  
Further Development

ABSTRACTDrugs that target both the liver and blood stages of malaria will be needed to reduce the disease's substantial worldwide morbidity and mortality. Evaluation of a 259-member library of compounds that block proliferation of the blood stage of malaria revealed several scaffolds—dihydroquinazolinones, phenyldiazenylpyridines, piperazinyl methyl quinolones, and bis-benzimidazoles—with promising activity against the liver stage. Focused structure-activity studies on the dihydroquinazolinone scaffold revealed several molecules with excellent potency against both blood and liver stages. One promising early lead with dual activity is 2-(p-bromophenyl)-3-(2-(diethylamino)ethyl)-2,3-dihydroquinazolin-4(1H)-one with 50% effective concentrations (EC50s) of 0.46 μM and 0.34 μM against liver stagePlasmodium bergheiANKA and blood stagePlasmodium falciparum3D7 parasites, respectively. Structure-activity relationships revealed that liver stage activity for this compound class requires a 3-dialkyl amino ethyl group and is abolished by substitution at theortho-position of the phenyl moiety. These compounds have minimal toxicity to mammalian cells and are thus attractive compounds for further development.

scholarly journals Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

2011 ◽  
Vol 77 (9) ◽  
pp. 2905-2915 ◽  
Author(s):  
Claire R. Shen ◽  
Ethan I. Lan ◽  
Yasumasa Dekishima ◽  
Antonino Baez ◽  
Kwang Myung Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Driving Forces ◽  
High Titer ◽  
Catalytic Efficiency ◽  
Anaerobic Growth ◽  
High Yield ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Content Type ◽  
Irreversible Reaction

ABSTRACT1-Butanol, an important chemical feedstock and advanced biofuel, is produced byClostridiumspecies. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway inEscherichia colito provide an irreversible reaction catalyzed bytrans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

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