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PubMed2005

An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH).

Fluorescence in situ hybridisation (FISH) is a common and popular method used to investigate microbial populations in natural and engineered environments. DNA oligonucleotide probes require accurate determination of the optimal experimental conditions for their use in FISH. Oligonucleotides targeting the rRNA of methanogenic Archaea at various taxonomic levels have previously been published, although when applied in FISH, no optimisation data has been presented. In this study, 3000 Euryarchaeota 16S rRNA gene sequences were phylogenetically analysed and previously published oligonucleotides were evaluated for target group accuracy. Where necessary, modifications were introduced or new probes were designed. The updated set of probes was optimised for use in FISH for a more accurate detection of methanogenic Archaea.

Archaea↗
PubMed2026

Phage therapy in oncology: opportunities for cancer prevention and treatment.

Bacteriophages (phages) are emerging as programmable biological therapeutics in oncology, extending beyond their traditional antimicrobial applications. This review proposes a phage-microbiome-immune-oncology axis that links microbial dynamics, immune modulation, and engineered phages to guide precision cancer prevention and therapy. Phages can eliminate cancer-associated bacteria, remodel the tumor microenvironment, enhance antitumor immunity, and deliver targeted therapeutic payloads. However, several critical challenges must be addressed to realize this therapeutic potential, particularly host immune responses that limit repeat dosing, inefficient tumor penetration, and the need for rigorous clinical validation. By examining phage-host-tumor interactions through robust model systems and highlighting translational opportunities, this review establishes phage therapy as a promising frontier in precision oncology that warrants accelerated clinical development.

Humans↗
PubMed2004

Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome.

Cell-free biology provides a unique opportunity to assess and to manipulate microbial systems by inverse metabolic engineering. We have applied this approach to amino acid metabolism, one of the systems in cell-free biology that limits protein synthesis reactions. Four amino acids (arginine, tryptophan, serine and cysteine) are depleted during a 3-h batch cell-free protein synthesis reaction under various conditions. By modifying the genome of the Escherichia coli strain used to make the cell extract, we see significant stabilization of arginine, tryptophan and serine. Cysteine, however, continues to be degraded. Cell-free protein synthesis with the modified cell extract produces increased yields of the cysteine-free protein Outer Membrane Protein T (OmpT).

Amino Acids↗
PubMed2004

Isolation and characterization of novel geldanamycin analogues.

New geldanamycin analogues with novel structures arising from direct microbial bioconversion and a genetically engineered geldanamycin producer were isolated and characterized. Three compounds, 15-hydroxygeldanamycin, a tricyclic geldanamycin analog (KOSN-1633), and methyl-geldanamycinate), were isolated after geldanamycin was added to a growing culture of the herbimycin producing strain-Streptomyces hygroscopicus AM-3672. Two related compounds, 17-formyl-17-demethoxy-18-O,-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-demethoxygeldanamycin were isolated from S. hygroscopicus NRRL 3602/pKOS279-78, a geldanamycin-producing strain containing various genes isolated from S. hygroscopicus AM-3672. Compared with geldanamycin, these five new compounds exhibited reduced cytotoxicity against SKBr3 cancer cells.

Antibiotics, Antineoplastic↗
PubMed1993

[Recombinant strains: their biological properties and the safety measures in working with them].

The review covers the problems of environmental safety of industrial microbial strains derived by genetic engineering techniques and used to prepare biologically active substances. E. coli was used as an example to show the significance of studying their biological characteristics, taxonomy, interaction with other microorganisms and macroorganisms. Based on the data available in the literature it is concluded that there is a need to work out a set of measures to continuously monitor recombinant microorganisms used in biotechnological industry.

Bacteria↗
PubMed2004

Dealing with complexity: evolutionary engineering and genome shuffling.

Comparative analysis of the growing number of microbial genome sequences has shown a high plasticity of genomes and several mechanisms for the adaptation of microbial cells to changing environmental conditions have been discovered. By contrast, the underlying metabolic networks of microorganisms are under strict control and relatively rigid, which poses a significant challenge for rational metabolic engineering approaches. Recursive shuffling of whole genomes has recently been demonstrated as an effective new evolutionary whole-cell engineering approach for the rapid improvement of industrially important microbial phenotypes.

Bacterial Proteins↗
PubMed2000

Engineering novel carotenoids in microorganisms.

A considerable number of microbial and plant carotenoid biosynthesis genes have been cloned over the past few years. Functional heterologous expression of most of these genes has made it possible to engineer carotenoid biosynthesis in non-carotenogenic E. coli and yeasts. Recently, gene combination and molecular breeding of pathways have been used to produce novel and rare high-value carotenoids.

Bacteria↗
PubMed1992

[Preparation and characteristics of protoplasts from various strains of Streptomyces roseoflavus var. roseofungini].

It was shown that the variants of the roseofungin-producing organism, which differ in their differentiation, antibiotic activity, structure and cell wall composition had different sensitivity to the protoplasting factors. The protoplasting increased the population heterogeneity: in strain 1128 it was evident from an increased frequency of the secondary colonies, in variant 1-68, folding of the colonies increased and their consistency become milder, sectorial colonies and colonies with coremia formed. It was in principle possible to transform the protoplasts of S. roseoflavus var. roseofungini by plasmid DNA, which suggests that the roseofungin-producing culture may be useful in genetic engineering.

Antifungal Agents↗
PubMed2026

A Comprehensive Review on the Biosynthesis of Tropane Alkaloids.

Tropane alkaloids (TA) constitute a class of plant specialized metabolites with important pharmaceutical applications, including the anticholinergic agents hyoscyamine and scopolamine and the local anesthetic cocaine. Over the past decade, advances in genomics, structural biology, and synthetic biology have substantially revised our understanding of TA biosynthesis, leading to the identification of numerous key biosynthetic enzymes and evolutionary mechanisms. This review comprehensively summarizes current knowledge of TA biosynthesis from precursor formation to structurally diverse end products. We describe the pathway from putrescine to tropinone, the stereoselective metabolic branching mediated by Tropinone Reductases, and the downstream biosynthesis of medicinal tropane alkaloids, calystegines, and cocaine. Particular emphasis is placed on recent discoveries concerning catalytic mechanisms, structural determinants of substrate specificity, metabolic compartmentalization, and the convergent evolution of TA biosynthesis in Solanaceae and Erythroxylaceae. We further integrate advances in genomics, evolutionary biology, and metabolic engineering to highlight emerging strategies for microbial production and pathway redesign. By providing a comprehensive synthesis of recent progress and critical perspectives on unresolved questions, this review offers an updated framework for understanding TA biosynthesis and supports future research in plant specialized metabolism, synthetic biology, and natural product engineering.

Tropanes↗
PubMed2004

Predicting the biodegradation products of perfluorinated chemicals using CATABOL.

Perfluorinated chemicals (PFCs) form a special category of organofluorine compounds with particularly useful and unique properties. Their large use over the past decades increased the interest in the study of their environmental fate. Fluorocarbons may have direct or indirect environmental impact through the products of their decomposition in the environment. It is a common knowledge that biodegradation is restricted within non-perfluorinated part of molecules: however, a number of studies showed that defluorination can readily occur during biotransformation. To evaluate the fate of PFCs in the environment a set of principal transformations was developed and implemented in the simulator of microbial degradation using the catabolite software engine (CATABOL). The simulator was used to generate metabolic pathways for 171 perfluorinated substances on Canada's domestic substances list. It was found that although the extent of biodegradation of parent compounds could reach 60%, persistent metabolites could be formed in significant quantities. During the microbial degradation a trend was observed where PFCs are transformed to more bioaccumulative and more toxic products. Perfluorooctanoic acid and perfluorooctanesulfonate were predicted to be the persistent biodegradation products of 17 and 27% of the perfluorinated sulphonic acid and carboxylic acid containing compounds, respectively.

Bacteria↗
PubMed2006

Metabolic engineering under uncertainty. I: framework development.

Standard bioprocess conditions have been widely applied for the microbial conversion of raw material to essential industrial products. Successful metabolic engineering (ME) strategies require a comprehensive framework to manage the complexity embedded in cellular metabolism, to explore the impacts of bioprocess conditions on the cellular responses, and to deal with the uncertainty of the physiochemical parameters. We have recently developed a computational and statistical framework that is based on Metabolic Control Analysis and uses a Monte Carlo method to simulate the uncertainty in the values of the system parameters [Wang, L., Birol, I., Hatzimanikatis, V., 2004. Metabolic control analysis under uncertainty: framework development and case studies. Biophys. J. 87(6), 3750-3763]. In this work, we generalize this framework to incorporate the central cellular processes, such as cell growth, and different bioprocess conditions, such as different types of bioreactors. The framework provides the mathematical basis for the quantification of the interactions between intracellular metabolism and extracellular conditions, and it is readily applicable to the identification of optimal ME targets for the improvement of industrial processes [Wang, L., Hatzimanikatis, V., 2005. Metabolic engineering under uncertainty. II: analysis of yeast metabolism. Submitted].

Bioreactors↗
PubMed1994

Survival and impact of genetically engineered Pseudomonas putida harboring mercury resistance gene in soil microcosms.

The survival of genetically engineered and wild-type Pseudomonas putida PpY101, that contained a recombinant plasmid pSR134 conferring mercury resistance, were monitored in andosol and sand microcosms. The survival of genetically engineered and wild-type P. putida was not significantly different in andosol. The population change of the two strains was dissimilar in andosol and sand. The survival of genetically engineered and wild-type P. putida strains was affected by the water content of andosol, and increased with the increment of the water content. The impact of the addition of genetically engineered and wild-type P. putida strains on indigenous bacteria and fungi was examined. Inoculation of both strains had no apparent effect on the density of indigenous microorganisms.

Drug Resistance, Microbial↗
PubMedDate not supplied

Soil microbial communities associated with Bt and non-Bt corn in three soils.

The effects of expression of Cry endotoxin by Bt corn (transgenic corn engineered to express Bacillus thuringiensis toxin) on soil microbial community structure were assessed in a growth chamber experiment. Two lines of transgenic corn expressing different Cry endotoxins were compared with their respective non-transgenic isolines in three soil types with differing textures. Phospholipid fatty acid (PLFA) profiles from bulk soil and community-level physiological profiles (CLPP) from the rhizosphere community were used to assess community structure. Differences in PLFA profiles due to soil type were significant, accounting for 73% of the total variability in the dataset. Differences in bacterial and fungal CLPP profiles due to soil type were statistically significant, but probably not biologically important, accounting for 6.3 and 3.8% of the total variability, respectively. Neither expression of Cry endotoxin nor corn line had a significant effect on microbial profiles, except in the high-clay soil where both factors significantly affected bacterial CLPP profiles (accounting for 6.6 and 6.1% of the variability in that soil, respectively). Expression of Cry endotoxin also significantly reduced the presence of eukaryotic PLFA biomarker in bulk soils, although it is unclear which groups of eukaryotes were affected. We conclude that the effects of transgenic Bt corn in this short-term experiment are small, and longer-term investigations are necessary.

Aluminum Silicates↗
PubMed2004

Substrate recognition by nonribosomal peptide synthetase multi-enzymes.

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is less clear for the thioesterase domains, which seem in certain cases to be dedicated to the hydrolysis/cyclization of their natural substrate, whereas in other cases they are largely permissive.

Models, Molecular↗
PubMed1998

A molecular view of microbial diversity in a dynamic landfill in Québec.

An Aroclor 1260 (polychlorinated biphenyl, PCB)-laden soil and one heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a secure, engineered landfill site in Québec were analyzed for microbial diversity using a clone library of the 16S rDNA sequences. Phylogenetic analysis revealed that three phyla and their major subdivisions of the domain Bacteria were highly represented in these samples despite the high pollution, particularly by PAHs. None of the 16S rDNA sequences obtained matched known sequences from cultivated bacterial species or from 16S rDNA sequences amplified directly from other environmental samples.

DNA, Bacterial↗
PubMed1993

Protein engineering of subtilisins to improve stability in detergent formulations.

Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

Amino Acid Sequence↗
PubMed2005

Suicidal genetically engineered microorganisms for bioremediation: need and perspectives.

In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.

Bacteria↗