scholarly journals Dihydrodipicolinate synthase from Thermotoga maritima

2006 ◽  
Vol 400 (2) ◽  
pp. 359-366 ◽  
Author(s):  
F. Grant Pearce ◽  
Matthew A. Perugini ◽  
Hannah J. Mckerchar ◽  
Juliet A. Gerrard
Keyword(s):  
Chemical Stability ◽  
Biosynthetic Pathway ◽  
Thermotoga Maritima ◽  
Sedimentation Coefficient ◽  
Thermophilic Bacterium ◽  
Lysine Biosynthesis ◽  
Dihydrodipicolinate Synthase ◽  
E Coli ◽  
High Level ◽  
Similar Kinetic

DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 °C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage.

scholarly journals Regulation of aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase in Lactobacillus plantarum

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Muhammad N. Cahyanto ◽  
Hiroko Kawasaki ◽  
Mariko Nagashio ◽  
Kazuhito Fujiyama ◽  
Tatsuji Seki
Keyword(s):  
Lactobacillus Plantarum ◽  
Lysine Biosynthesis ◽  
Dihydrodipicolinate Synthase ◽  
Homoserine Dehydrogenase ◽  
Biosynthetic Genes ◽  
E Coli ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
Reductase Gene

The use of a lysine-overproducing strain of Lactobacillus plantarum in food or feed fermentations may lead to the production of lysine-rich products. The availability of functional genes and information on the regulation of lysine biosynthesis are required to develop a lysine-overproducing strain. The genome sequence of L. plantarum revealed putative lysine biosynthetic genes, some of which may produce isozymes. This study examined the functionality of the genes and the regulation of the first four enzymes of lysine biosynthesis, together with homoserine dehydrogenase, in L. plantarum. The genes were expressed in Escherichia coli, and the regulation of the enzymes was studied in cell extracts of both recombinant E. coli and L. plantarum. Among seven lysine biosynthetic genes studied (aspartokinase genes, thrA1 and thrA2; aspartate semialdehyde dehydrogenase genes, asd1 and asd2; dihydrodipicolinate synthase genes, dapA1 and dapA2; and the dihydrodipicolinate reductase gene, dapB) plus two homoserine dehydrogenase genes (hom1 and hom2), the products of six genes, i.e. thrA2, asd2, dapA1, dapB, hom1 and hom2, showed obvious enzyme activities in vitro. The product of one of the homoserine dehydrogenase genes, hom1, exhibited both homoserine dehydrogenase and aspartokinase activities. However, the aspartokinase activity was mainly due to ThrA2 and was inhibited by l-lysine and repressed by l-threonine, and the homoserine dehydrogenase activity was mainly due to Hom2 and was inhibited by l-threonine. The aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase were not regulated by the end-products of the pathway.

scholarly journals Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

2016 ◽  
Vol 113 (12) ◽  
pp. 3209-3214 ◽  
Author(s):  
Bradley Walters Biggs ◽  
Chin Giaw Lim ◽  
Kristen Sagliani ◽  
Smriti Shankar ◽  
Gregory Stephanopoulos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Complex Molecules ◽  
E Coli ◽  
Precursor Synthesis ◽  
P450 Expression ◽  
High Level

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

2019 ◽  
Vol 35 (6) ◽  
pp. 67-72 ◽  
Author(s):  
I.V. Manukhov ◽  
L.S. Yaguzhinsky ◽  
M.V. Bermeshev ◽  
M.A. Zisman ◽  
V.G. Pevgov ◽  
...  
Keyword(s):  
Toxic Effect ◽  
Atmospheric Oxygen ◽  
Inducible Promoter ◽  
Oxygen Species ◽  
Bacterial Cells ◽  
Unique Identifier ◽  
E Coli ◽  
Lux Biosensors ◽  
High Level ◽  
Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

High-Level Expression of Human β-Defensin-2 Gene with Rare Codons in E. coli Cell-Free System

2006 ◽  
Vol 13 (2) ◽  
pp. 155-161 ◽  
Author(s):  
Haiqin Chen ◽  
Zhinan Xu ◽  
Naizheng Xu ◽  
Peilin Cen
Keyword(s):  
Coli Cell ◽  
High Level Expression ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Rare Codons ◽  
High Level

scholarly journals Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

2006 ◽  
Vol 400 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Eric Di Luccio ◽  
Robert A. Elling ◽  
David K. Wilson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Thermotoga Maritima ◽  
Xylose Reductase ◽  
Sulfolobus Solfataricus ◽  
Sequence Comparisons ◽  
E Coli ◽  
Fluorescence Measurements ◽  
Dual Specificity ◽  
Substrate Dependence

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

scholarly journals Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5391-5397 ◽  
Author(s):  
Si Jae Park ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Biosynthetic Pathway ◽  
Pha Synthase ◽  
Enzymatic Analysis ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Enoyl Coa Hydratase

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

scholarly journals Molecular survey of mcr1 and mcr2 plasmid mediated colistin resistance genes in Escherichia coli isolates of animal origin in Iran

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kayhan Ilbeigi ◽  
Mahdi Askari Badouei ◽  
Hossein Vaezi ◽  
Hassan Zaheri ◽  
Sina Aghasharif ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Companion Animals ◽  
Multidrug Resistant ◽  
Animal Origin ◽  
Colistin Resistance ◽  
E Coli ◽  
High Level ◽  
Farming Industry

Abstract Objectives The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is one of the major public health concerns as colistin is the last-resort antibiotic for treating infections caused by multidrug-resistant Gram-negative bacteria. We aimed to determine the prevalence of the prototype widespread colistin resistance genes (mcr-1 and mcr-2) among commensal and pathogenic Escherichia coli strains isolated from food-producing and companion animals in Iran. Results A total of 607 E. coli isolates which were previously collected from different animal sources between 2008 and 2016 used to uncover the possible presence of plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) by PCR. Overall, our results could not confirm the presence of any mcr-1 or mcr-2 positive E. coli among the studied isolates. It is concluded that despite the important role of food-producing animals in transferring the antibiotic resistance, they were not the main source for carriage of mcr-1 and mcr-2 in Iran until 2016. This study suggests that the other mcr variants (mcr-3 to mcr-9) might be responsible for conferring colistin resistance in animal isolates in Iran. The possible linkage between pig farming industry and high level of mcr carriage in some countries needs to be clarified in future prospective studies.

scholarly journals Elucidation of the d ‐lysine biosynthetic pathway in the hyperthermophile Thermotoga maritima

FEBS Journal ◽  
2018 ◽  
Vol 286 (3) ◽  
pp. 601-614 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Masumi Katane ◽  
Yasuaki Saitoh ◽  
Masae Sekine ◽  
Hiroshi Homma
Keyword(s):  
Biosynthetic Pathway ◽  
Thermotoga Maritima

Fate of Escherichia coli O157:H7 and Other Coliforms in Commercial Mayonnaise and Refrigerated Salad Dressing

1995 ◽  
Vol 58 (1) ◽  
pp. 13-18 ◽  
Author(s):  
ERROL V. RAGHUBEER ◽  
JIM S. KE ◽  
MICHAEL L. CAMPBELL ◽  
RICHARD S. MEYER
Keyword(s):  
Escherichia Coli ◽  
Storage Temperature ◽  
Enterobacter Aerogenes ◽  
Antimicrobial Effect ◽  
Coliform Bacteria ◽  
Escherichia Coli O157 ◽  
Salad Dressing ◽  
E Coli ◽  
Survival Pattern ◽  
High Level

Commercial mayonnaise and refrigerated ranch salad dressing were inoculated at two levels with two strains of Escherichia coli O157:H7, a non-pathogenic E. coli, and the non-fecal coliform Enterobacter aerogenes. Results showed that at the high inoculation level (>106 colony forming units [CFU]/g) in mayonnaise stored at room temperature (ca. 22°C) both strains of O157:H7 were undetected at 96 h. At the high inoculation level, all strains of coliform bacteria tested survived longer in salad dressing stored at 4°C than in mayonnaise stored at 22°C. The O157:H7 strains were still present at low levels after 17 days. The survival time in the low-level inoculum (104CFU/g) study decreased, but the survival pattern in the two products was similar to that observed in the high-level inoculum study. Slight differences in survival among strains were observed. The greater antimicrobial effect of mayonnaise may be attributable to differences in pH, water activity (aw), nutrients, storage temperature, and the presence of lysozyme in the whole eggs used in the production of commercial mayonnaise. Coliform bacteria survived longer in refrigerated salad dressing than in mayonnaise particularly at the high-level inoculum. Both mayonnaise (pH 3.91) and salad dressing (pH 4.51) did not support the growth of any of the microorganisms even though survival was observed.

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