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At least 811 records · Page 45Linked to original sources
PubMed1998

Technological advances in high-throughput screening.

A variety of assay technologies continue to be developed for high-throughput screening. These include cell-based assays, surrogate systems using microbial cells such as yeast and bacterial two-hybrid and three-hybrid systems, and systems to measure nucleic acid-protein and receptor-ligand interactions. Modifications have been developed for cell-free, homogeneous assay systems, such as time-resolved fluorescence, fluorescence polarization and the scintillation proximity assay. Innovations in engineering and chemistry have led to delivery systems for nanoliter volumes and sensitive biosensors for ultra-high-throughout screening conducted in nanoliter and picoliter volumes. Spectroscopic methods have been extended to read single molecule fluorescence. Technologies are being developed to identify new targets from genomic information in order to design the next generation of screens.

Calmodulin↗
PubMed2006

Enzymatic cross-linking versus radical polymerization in the preparation of gelatin PolyHIPEs and their performance as scaffolds in the culture of hepatocytes.

Highly open porous biodegradable scaffolds, based on gelatin A3, were fabricated with the aim of using them for tissue-engineering applications. The fabrication process is based on an emulsion-templating technique. In the preparation of gelatin scaffolds two different cross-linking procedures were adopted: (I) radical polymerization of the methacrylate functionalities, previously introduced onto the gelatin chains and (II) formation of isopeptide bridges among the gelatin chains promoted by the enzyme microbial transglutaminase. The method of cross-linking exerts a pronounced effect on the morphology of the porous biomaterials: radical polymerization of methacrylated gelatin allowed the production of scaffolds with a better defined porous structure, while the enzymatically cross-linked scaffolds were characterized by a thinner skeletal framework. A suitable sample of each kind of the differently cross-linked porous biomaterials was tested for the culture of hepatocytes. The scaffold obtained by radical polymerization possessed a morphology characterized by relatively large voids and interconnects, and as a consequence, it was more suitable for hepatocytes colonization. On the other hand, the enzymatically cross-linked scaffold resulted in less cytotoxicity and the cultured hepatocytes expressed a better differentiated phenotype, as evidenced by a greater expression and more correct localization of key adhesion proteins.

Animals↗
PubMed2025

Engineering recombination machinery facilitates the construction of yeast cell factories.

Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.

Metabolic Engineering↗
PubMed2025

Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate.

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC-MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner-Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.IMPORTANCEBiomass pretreatment processes release fermentable sugars from lignocellulosic biomass, but they also generate inhibitors that can impact microbial metabolism. This study provides a systems-level evaluation of how Zymomonas mobilis responds to hydrolysate stress, revealing distinct physiological and lipid membrane remodeling responses. While some stress responses overlap with those induced by ethanol and isobutanol toxicity, both valuable biofuels, hydrolysate exposure elicits unique metabolic shifts. These findings offer valuable insights for engineering Z. mobilis strains with improved tolerance and performance for efficient bioconversion of lignocellulosic hydrolysates into biofuels and bioproducts.

Zymomonas↗
PubMed1996

A stringently controlled expression system for analysing lateral gene transfer between bacteria.

The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems. We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient-specific phenotype. The system consists of an engineered lacZ (encoding beta-galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter-operator from lambda bacteriophage and the 5' non-coding leader region of the inp gene encoding the IS 10 transposase function. Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans-acting repressors working at the level of transcription (the Cl-EK117 protein from the lambda phage), and at the level of translation (the antisense RNA-OUT of the IS 10 element). When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed. The system was validated with Pseudo-monas putida, and conjugational transfer frequencies of the reporter element as low as 10(-6) were detected. The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real-time in situ detection, will facilitate the monitor-ing of gene flow in a non-disruptive manner within the environment.

Bacteria↗
PubMed2004

[Application of bioinformatics in researches of industrial biocatalysis].

Industrial biocatalysis is currently attracting much attention to rebuild or substitute traditional producing process of chemicals and drugs. One of key focuses in industrial biocatalysis is biocatalyst, which is usually one kind of microbial enzyme. In the recent, new technologies of bioinformatics have played and will continue to play more and more significant roles in researches of industrial biocatalysis in response to the waves of genomic revolution. One of the key applications of bioinformatics in biocatalysis is the discovery and identification of the new biocatalyst through advanced DNA and protein sequence search, comparison and analyses in Internet database using different algorithm and software. The unknown genes of microbial enzymes can also be simply harvested by primer design on the basis of bioinformatics analyses. The other key applications of bioinformatics in biocatalysis are the modification and improvement of existing industrial biocatalyst. In this aspect, bioinformatics is of great importance in both rational design and directed evolution of microbial enzymes. Based on the successful prediction of tertiary structures of enzymes using the tool of bioinformatics, the undermentioned experiments, i.e. site-directed mutagenesis, fusion protein construction, DNA family shuffling and saturation mutagenesis, etc, are usually of very high efficiency. On all accounts, bioinformatics will be an essential tool for either biologist or biological engineer in the future researches of industrial biocatalysis, due to its significant function in guiding and quickening the step of discovery and/or improvement of novel biocatalysts.

Biocatalysis↗
PubMed1994

New composite biocarriers engineered to contain adsorptive and ion-exchange properties improve immobilized-cell bioreactor process dependability.

Novel biocarriers that combine the adsorptive properties of activated carbon with the ion-exchange properties of zeolite-based type Z inorganic oxide biocarriers (D. R. Durham, L. C. Marshall, J. G. Miller, and A. B. Chmurny, Appl. Environ, Microbiol. 60:3329-3335, 1994.) were developed. These biocarriers, designated Type CZ, possess fundamental properties that heretofore have not been described for available microbial immobilization matrices. Type CZ biocarriers provide an environment that promotes dense microbial colonization and maintains bioreactor productivity by buffering immobilized microorganisms from unfavorable operating conditions. Data demonstrating protection of immobilized bacteria from organic shock loads and extended pH shocks are presented. In addition, bioreactors containing the composite Type CZ biocarriers continue to remove waste stream contaminants during periods of oxygen deprivation and nutrient limitation.

Journal Article↗
PubMed2005

Strain-controlled enzymatic cleavage of collagen in loaded matrix.

The purpose of this investigation is to support the novel hypothesis that collagenous matrices are intrinsically "smart" load-adapting biomaterials. This hypothesis is based fundamentally on the postulate that tensile strain directly modulates the susceptibility of collagen molecules to enzymatic degradation (i.e., protects molecules which are under load from cleavage). To test this postulate, collagenase (Clostridiopeptidase A) was applied to a uniaxially loaded, anisotropic, devitalized, collagenous matrix in which a subset of fibrils was loaded in tension while the remaining fibrils carried little or no load. The collagen degradation pattern (as assessed by polarization and transmission electron microscopy) was found to correspond inversely to the tensile stress field such that fibrils under lower tensile load were preferentially cleaved. These results have immediate implications for tissue engineering of load-bearing collagenous matrices in vitro and may contribute significantly to our understanding of synthesis, remodelling, and pathogenesis of collagen matrices in vivo.

Catalysis↗
PubMed2005

Use of terminal tap water filter systems for prevention of nosocomial legionellosis.

Hospital water supplies often contain Legionella spp. and represent a potential source of nosocomial infection, especially for immunocompromised patients or those in intensive care units. Therefore, pathogen-free water should be provided for such high-risk patients. Surveillance of splash water was performed in high-risk patient care areas at Berlin Charité-University Medicine (506 samples) and Medical School Hannover (767 samples) to investigate the ability to provide water that was free from Legionella spp. by the use of disposable, terminal tap water filter systems with non-impregnated, as well as impregnated, filters with prolonged usage intervals. Twenty (Berlin) and 32 (Hannover) water outlets were provided with disposable filters with a pore size of 0.2 microm. Testing of unfiltered tap water revealed growth of Legionella spp. in 53 of 210 (Berlin) and 30 of 32 (Hannover) samples. Non-impregnated, terminal, disposable water filters at taps used for high-risk patient care led to water free from Legionella spp. in 154 of 155 (99.4%) samples after three to four days and in 137 of 141 (97.2%) samples after six to seven days. When testing a new impregnated filter, 255 of 256 (99.6%) samples remained free from Legionella spp. after continuous use for seven days, as recommended by the manufacturers, and also after 10 days. Samples that were positive for Legionella spp. contained 1-4 cfu/mL. We believe that an impregnated filter system is suitable for the prevention of nosocomial Legionellosis in high-risk patient care areas.

Berlin↗
PubMed2006

Identifying pioneer bacterial species responsible for biofouling membrane bioreactors.

More effective control of membrane biofouling in membrane bioreactors (MBRs) lies in the fundamental understanding of the pioneer microorganisms responsible for surface colonization that leads to biofilm formation. In this study, the composition of the planktonic and sessile microbial communities inhabiting four laboratory-scale MBR systems were compared using amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA gene sequencing. The ARDRA results suggest that the microbial communities on membrane surfaces could be very different from the ones in the suspended biomass. Phylogenetic analysis based on the 16S rRNA gene sequences provided a list of bacteria that might be the pioneers of surface colonization on microfiltration membranes. The results further suggested that research on the mechanisms of cell attachment in such an engineering environment could be critical for future development of appropriate biofouling control strategies.

Bacteria↗
PubMed2005

Functional ingredient production: application of global metabolic models.

The biotechnology industry continuously explores new ways to improve the performance of microbial strains in fermentation processes. Recent focus has been on new genome-wide modelling approaches in functional genomics, which aim to take full advantage of genome sequence data, transcription profiling, proteomics and metabolite profiling for strain improvement. The integration of global metabolic models with genetic and regulatory models will be essential for the practice of metabolic engineering for strain improvement to move forward, simply because we cannot rely on our intuition to grasp the complexity of the biological systems involved.

Bacterial Physiological Phenomena↗
PubMed2001

A 3.1-kb genomic fragment of Bacillus subtilis encodes the protein inhibiting growth of Xanthomonas oryzae pv. oryzae.

AIMS: To clone genes of Bacillus subtilis encoding peptides that inhibit the growth of Xanthomonas orzae pv. oryzae (Xoo). METHODS AND RESULTS: A 3.1-kb DNA fragment from B. subtilis SO113 encoding peptides that inhibit the growth of Xoo (anti-Xoo, showing an inhibition zone) was isolated from a plasmid library of B. subtilis 6 GM15. Sequence analysis revealed that it contained three complete open reading frames (ORFs): ybcO, ybcS and a novel ORF designated ybcPQ. Deleting the last 96 bp of ybcS from the plasmid eliminated the anti-Xoo activity, suggesting that ybcS is required for producing the anti-Xoo activity. However, no anti-Xoo activity could be detected for the plasmid with ybcS alone. Further analysis showed that ybcO, at least, was also required to obtain the anti-Xoo activity. CONCLUSIONS: A fragment of B. subtilis has been cloned that expresses an anti-Xoo activity that requires ybcS and ybcO. SIGNIFICANCE AND IMPACT OF THE STUDY: These genes could be useful for the genetic engineering of resistance to rice bacterial diseases and for the design of new anti-Xoo biocontrol agents.

Amino Acid Sequence↗
PubMed2005

Development of a rapid assay for determining the relative abundance of bacteria.

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10(3) to 10(4) and 10(4) to 10(5) bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.

Bacteria↗
PubMedDate not supplied

Microbial production of L-lysine: a review.

Microbial production of L-lysine has been reviewed with 251 references. The review includes different lysine producing microorganisms, their optimum cultural conditions, yield, assay and process of product recovery. It includes a discussion on the pathway of aspartate family of amino acid's biosynthesis in bacteria and its regulation. Achievements in this regard made through genetic engineering have also been included.

Bacteria↗
PubMed2005

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity.

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (mu(a)) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml(-1) and 0.9-2.1 g l(-1) dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm(3) was observed in cultures supplemented with 3 g l(-1) xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the mu(a). Counter-intuitively, an increase in the mu(a) within the experimental range examined resulted in an increase in the rate of gas-liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.

Culture Media↗
PubMedDate not supplied

[Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain].

The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B. subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model. As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants. The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

Animals↗
PubMed1994

Using feed-forward neural networks for estimation of microbial concentration in a simulated biochemical process.

This work investigated the feasibility of using feed-forward neural networks for estimation of a state variable in a process with highly non-linear characteristics. A biochemical process was considered where the microorganism Saccharomyces cerevisiae, a yeast, grows in a chemostat on a glucose substrate and produces ethanol as a product of primary energy metabolism. Three state variables for the process are the microbial concentration, substrate concentration and product concentration. The Levenberg-Marquardt Method was used to train the neural networks by minimising the sum of squares of the residuals. The inputs to the networks were the measured variable (product concentration) and the control variable (dilution rate). The output of the network was an estimate for the microbial concentration. Earlier work had shown that system identification of this biochemical process could be performed quite well using feed-forward neural networks. This work demonstrated that state estimation can also be performed successfully using feed-forward neural networks. Knowledge of the process model is not required. The method is simple, reliable and accurate enough for engineering purposes. It can save a lot of expense on sensors, their installation and maintenance.

Neural Networks, Computer↗