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PubMedDate not supplied

Application of mathematical tools for metabolic design of microbial ethanol production.

Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates.

Bacteriological Techniques↗
PubMed2006

Manufacturing of recombinant therapeutic proteins in microbial systems.

Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.

Antibodies, Monoclonal↗
PubMed2001

Comparison of methods for determination of microbial biomass in wastewater.

Microbial biomass in wastewater was determined by methods used in environmental microbiology and by a method used in wastewater engineering based on a conceptual model simulating fundamental microbial processes in wastewater from measured oxygen uptake rates. The methods originating from environmental microbiology are based on staining and counting of cells for the determination of total cell biomass (acridine orange and DAPI), physiological state of cells (LIVE/DEAD BacLight) and activity of cells (reduction of the redox dye CTC and microautoradiography). Depending on the staining method applied, cell biomasses yielded 15-86% of the biomass defined by the model, and good correlations between cell biomass and model biomass were found. Cell biomass, oxygen uptake and acetate uptake were measured in wastewater, where acetate was added. Substrate uptake rates were found not to be proportional to the increases in cell biomass, suggesting that only a small fraction of the cell biomass was responsible for the main part of the substrate uptake. Despite the differences found between cell biomass and model biomass, it was recommended to use the conceptual model as an engineering tool for simulation of microbial processes and wastewater quality changes. However, there should be a clear distinction between the terms 'model biomass', 'cell biomass' and different activity measurements of cells.

Bacteria↗
PubMed2003

Cyanobacterial postgenomic research and systems biology.

The genomic era brought with it the capacity to unlock complex interactions in organisms and biological systems. Currently, by exploiting genomic and associated protein information through in silico analyses, postgenomic research is developing rapidly. This field, which encompasses functional genomics, structural genomics, transcriptomics, pharmacogenomics, proteomics and metabolomics, allows for a systems-wide approach to biological studies. To date, bacterial postgenomic research has focused mainly on a few representative pathogenic species, leaving the vast majority of the microbial community relatively overlooked. Amongst the under-represented microorganisms are the cyanobacteria, which are important for their beneficial natural product production, bioremediation and energy applications. Here, we highlight the current status of cyanobacterial postgenomic research and assess the potential for future metabolic engineering and "cell factory" or "microbial cell" development.

Biotechnology↗
PubMed2026

From dysbiosis to resilience: Microbiome engineering for sustainable shrimp aquaculture.

The intensification of shrimp aquaculture has increased exposure to disease, environmental perturbations, and antimicrobial pressure, making microbial stability increasingly relevant to sustainable production. Microbiome stability-encompassing resistance to disturbance and resilience of functional recovery-provides an ecological framework for understanding how shrimp and culture-environment microbial communities respond to intensive farming. This review examines the transition from microbial homeostasis to dysbiosis and evaluates how microbiome engineering could redirect disrupted communities towards resilient states. Evidence is integrated across the intestine, hepatopancreas, rearing water, sediment and biofloc to assess how host genetics, ontogeny, diet, culture conditions, antibiotics and pollutants shape microbiome assembly and destabilization. Disease-associated changes in acute hepatopancreatic necrosis disease, white faeces syndrome, Enterocytozoon hepatopenaei infection, and white spot syndrome virus infection are critically evaluated, with explicit separation of associations, pathogen-induced dysbiosis, and community-level causality. Established and emerging interventions-including probiotics, prebiotics, synbiotics, functional diets, biofloc management, phages, postbiotics, microbiota transplantation and synthetic microbial communities-are assessed according to their capacity to modify microbial function, persistence and recovery rather than taxonomic change alone. We further examine how multi-omics, microbiome-informed breeding, and environmental monitoring could support biomarker development, predictive decision support and context-specific intervention. We argue that progress requires a shift from taxonomic description to function-guided engineering, from endpoint comparisons to direct measurement of resilience, and from laboratory efficacy to reproducible farm-scale validation. Overall, microbiome management may contribute to more disease-resilient and sustainable shrimp production, provided that its effectiveness can be validated under commercial farming conditions.

Dysbiosis↗
PubMed1983

Anaerobic fermentations--some new possibilities.

Anaerobic fermentations start with the major advantage that, unlike aerobic processes, they do not incur the cost penalties associated with the need to sustain high rates of culture aeration, agitation and cooling. Even so, many of the more traditional fermentations that yield high volume, low value products (such as alcohols or fatty acids) are currently only economically viable if they utilize biological wastes and agricultural surpluses as substrates. To achieve maximum conversion efficiency it is necessary to select the appropriate species/strain of micro-organism which will perform best under the intended conditions of operation (which could include elevated temperatures and extremes of pH). Knowledge of the mechanisms whereby the fermentation is physiologically controlled can then suggest means, including genetically engineered strain improvements and/or the establishment of stable co-cultures with other microbes, whereby the yield of a desired product can be optimized. The biology of a fermentation process can thus be rendered sufficiently consistent and predictable for the biochemical engineer to be able to achieve optimal fermenter productivity. Anaerobic processes are particularly well suited to a continuous flow mode of operation (with cell retention) and new techniques for selective product removal and concentration can reduce the energy costs associated with 'downstream processing'. Hitherto, most interest has been centred on those anaerobic fermentations of renewable biomass which yield immediately useful products (fuel alcohols, methane). However, by combining biological and chemical procedures the profitability of even some superficially unpromising fermentations could be substantially enhanced. Collaborative research by microbial physiologists, geneticists and biochemical engineers is a prerequisite for marketable success in new applications of fermentative anaerobes and their enzymes.

Anaerobiosis↗
PubMed1998

Surface plasmon resonance biosensors as a tool in antibody engineering.

Modern gene technology combined with efficient microbial expression systems provides tools to produce antibodies with reduced functional size and improved binding properties as well as antibody fusions or novel antibodies. Surface plasmon resonance based biosensors, which measure antigen-antibody interactions in real-time, can be used for a diverse characterization of the modified antibodies. To date, the majority of published work originates from real-time biospecific interaction analysis based on the BIAcore instruments. This article describes the range of applications in antibody engineering in which BIAcore has been applied.

Animals↗
PubMed2000

Enzymes involved in the aerobic bacterial degradation of N-heteroaromatic compounds: molybdenum hydroxylases and ring-opening 2,4-dioxygenases.

Many N-heteroaromatic compounds are utilized by micro-organisms as a source of carbon (and nitrogen) and energy. The aerobic bacterial degradation of these growth substrates frequently involves several hydroxylation steps and subsequent dioxygenolytic cleavage of (di)hydroxy-substituted heteroaromatic intermediates to aliphatic metabolites which finally are channeled into central metabolic pathways. As a rule, the initial bacterial hydroxylation of a N-heteroaromatic compound is catalyzed by a molybdenum hydroxylase, which uses a water molecule as source of the incorporated oxygen. The enzyme's redox-active centers - the active site molybdenum ion coordinated to a distinct pyranopterin cofactor, two different [2Fe2S] centers, and in most cases, flavin adenine dinucleotide - transfer electrons from the N-heterocyclic substrate to an electron acceptor, which for many molybdenum hydroxylases is still unknown. Ring-opening 2,4-dioxygenases involved in the bacterial degradation of quinaldine and 1H-4-oxoquinoline catalyze the cleavage of two carbon-carbon bonds with concomitant formation of carbon monoxide. Since they contain neither a metal center nor an organic cofactor, and since they do not show any sequence similarity to known oxygenases, these unique dioxygenases form a separate enzyme family. Quite surprisingly, however, they appear to be structurally and mechanistically related to enzymes of the alpha/beta hydrolase fold superfamily. Microbial enzymes are a great resource for biotechnological applications. Microbial strains or their enzymes may be used for degradative (bioremediation) or synthetic (biotransformation) purposes. Modern bioremediation or biotransformation strategies may even involve microbial catalysts or strains designed by protein engineering or pathway engineering. Prerequisite for developing such modern tools of biotechnology is a comprehensive understanding of microbial metabolic pathways, of the structure and function of enzymes, and of the molecular mechanisms of biocatalysis.

Bacteria, Aerobic↗
PubMed2026

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗
PubMed2006

Effects of endogenous substrates on adaptation of anaerobic microbial communities to 3-chlorobenzoate.

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.

Adaptation, Physiological↗
PubMed2006

Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli.

Nonribosomal peptides (NRPs) are a class of microbial secondary metabolites that have a wide variety of medicinally important biological activities, such as antibiotic (vancomycin), immunosuppressive (cyclosporin A), antiviral (luzopeptin A) and antitumor (echinomycin and triostin A) activities. However, many microbes are not amenable to cultivation and require time-consuming empirical optimization of incubation conditions for mass production of desired secondary metabolites for clinical and commercial use. Therefore, a fast, simple system for heterologous production of natural products is much desired. Here we show the first example of the de novo total biosynthesis of biologically active forms of heterologous NRPs in Escherichia coli. Our system can serve not only as an effective and flexible platform for large-scale preparation of natural products from simple carbon and nitrogen sources, but also as a general tool for detailed characterizations and rapid engineering of biosynthetic pathways for microbial syntheses of novel compounds and their analogs.

Antineoplastic Agents↗
PubMed1991

Cellular engineering.

Cellular engineering applies the principles and methods of engineering to the problems of cell and molecular biology of both a basic and applied nature. As biomedical engineering has shifted from the organ and tissue level to the cellular and sub-cellular level, cellular engineering has emerged as a new area. A cornerstone of much of this activity is cell culture technology, i.e., the ability to grow living cells in the artificial environment of a laboratory. Cellular engineering includes the role of engineering in both basic cell biology research and in the making of products which use living cells, e.g., tissue engineering and bioprocess engineering. The former involves the use of living cells in the development of biological substitutes for the restoration or replacement of function, and the latter the use of living cells to manufacture a biochemical product, e.g., through the use of recombinant DNA technology. In fact, as biomedical engineering has expanded to include the cellular level, and bioprocess engineering has shifted in interest from microbial organisms to include mammalian cells, there are intellectual issues in which an interest is shared by these two formerly separate areas of engineering activity. Cellular engineering thus transcends the field of biomedical engineering.

Biomedical Engineering↗
PubMedDate not supplied

Microbial sensor for new-generation cephalosporins based in a protein-engineered beta-lactamase.

A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.

Biosensing Techniques↗
PubMed2026

Metabolic engineering of Candida yeasts for biotechnological applications.

Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.

Candida↗
PubMed2026

Advances in the Application of Adenine Base Editor (ABE) in Biology and Medicine: Prospects and Challenges.

Adenine base editors (ABEs), which achieve A·T to G·C conversions in the genome precisely, symbolize a groundbreaking development in genetic engineering across animal, plant, and microbial systems. This review systematically summed up the research progress and current challenges of ABE in medical and biological applications: it outlined the historical context and pivotal milestones of its technological development; it emphasized major therapeutic advances for genetic diseases including spinal muscular atrophy, mitochondrial genetic disorders, and hyperlipidemia; it provided a comprehensive overview of its prospective uses for enhancing genetic traits in agricultural crops, including grains and fruits; this review conducted a multidimensional assessment of ABE performance through systematic comparison with other base editing technologies, comprehensively evaluating both editing efficiency and inherent limitations. It specifically addresses biosecurity risks such as off-target effects and genomic instability. Finally, safety concerns were proposed as the central challenge hindering its clinical translation, although ABE holds immense promise for precision medicine and agricultural breeding. Unlike previous reviews that mainly summarized early ABE development and general applications, this review particularly emphasizes recently engineered ABE systems, translational bottlenecks, delivery strategies, comparative clinical feasibility, and unresolved biosafety challenges that currently limit broader therapeutic and agricultural applications.

Adenine base editors↗
PubMed1995

Microbial biofilms and biofilm reactors.

Scientists and engineers have realized the industrial and environmental significance of biofilm accumulation and activity. The ability to predict and control biofilm formation has led to less fouling and corrosion in industrial systems and a better understanding of biofilm importance in natural aquatic systems. Understanding the fundamental processes contributing to biofilm formation is beneficial to anyone involved with natural or industrial systems where biofilms may play a significant role in determining variables such as bulk water quality, toxic compound biodegradation, or product quality.

Adsorption↗
PubMed1992

Microbial bioconversion of pollutants.

Microorganisms totally detoxicate xenobiotics of various chemical structures, which are serious and, in some cases, very hazardous pollutants. At present, the efforts of a number of researchers promoted the establishment in this country of a collection of microorganisms able to degrade volatile toxic pollutants--toluene, isomeric xylenes, styrene, alpha-methylstyrene, crotonaldehyde; widely distributed xenobiotics chlorobenzoic acids; isomeric aryldicarboxylic acids; and ecologically hazardous pollutants such as aromatic nitrocompounds. The active strains-destructors are mainly representatives of the genera Pseudomonas and Rhodococcus. Research into their physiological characteristics, key enzymes, pathways of xenobiotics degradation, genetic mechanisms determining the degradation of these foreign compounds, and behaviour of the strains in a real environment made it possible to develop the theoretical principles of using these microbial cultures to purify real industrial wastes and remediate polluted areas of soil and water. Improvement of the methods of immobilizing the active xenobiotics-degrading strains on cheap and efficient carriers made it possible to significantly intensify the cleanup process of industrial wastes and eliminate a number of problems during the development of the biotechnologies for industrial waste cleanup. Successfully operated at present are the biotechnologies of the local cleanup of waste waters of terephthalate production, microbial purification of industrial waste waters in nylon-66 production from hexamethylenediamine, purification of coke production wastes from phenols, waste waters of polyisocyanate production from aromatic amines, local purification of waste waters in synthetic rubber production from alpha-methylstyrene, acetaldehyde production wastes from crotonaldehyde and mercury. Microbial strains constructed by gene engineering methods for the cleanup of contaminated soils from dicofol and 3-chlorobenzoate were successfully applied (Golovleva et al. 1988).

Biodegradation, Environmental↗