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At least 109 records · Page 6Linked to original sources

PubMed2000

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris.

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1-101 of the human GAD67 protein fused to amino acids 96-585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.

Journal Article↗

PubMed2000

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Journal Article↗

PubMed2002

Nutraceutical production with food-grade microorganisms.

Over the past few years a number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. These include components that have a proven beneficial effect on human health, such as low-calorie sugars and B vitamins. Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food-grade microoganisms means that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.

Antioxidants↗

PubMed1997

Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content.

Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable advances have been made in the construction of inducible gene expression systems based on the capacity of these bacteria to utilize specific sugars or to secrete autoinducing peptides that are involved in quorum sensing. These controlled expression systems allow for present and future exploitation of these bacteria as cell factories in medical, agricultural, and food biotechnology.

Biotechnology↗

PubMed1999

Genomics for food biotechnology: prospects of the use of high-throughput technologies for the improvement of food microorganisms.

Functional genomics is currently the most effective approach for increasing the knowledge at the molecular level of metabolic and adaptive processes in whole cells. High-throughput technologies, such as DNA microarrays, and improved two-dimensional electrophoresis methods combined with tandem mass-spectroscopy, supported by bioinformatics, are useful tools for food biotechnology, which depends on detailed knowledge of the properties of food microbes (and pathogens) in their industrial, food and consumer environments. Genomics of food microbes, based on rapidly emerging genome sequence information, generates valuable knowledge that can be used for metabolic engineering, improving cell factories and development of novel preservation methods. Furthermore, pre- and probiotic studies, characterization of stress responses, studies of microbial ecology and, last but not least, development of novel risk assessment procedures will be facilitated.

Bacillus subtilis↗

PubMed2001

The expanding universe of alkaloid biosynthesis.

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

Acridines↗

PubMed1999

Gene expression systems for lactic acid bacteria.

Considerable advances have been made in the genetics and molecular biology of lactic acid bacteria, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus spp. These have resulted in the construction of constitutive gene expression cassettes, inducible gene expression systems, and specific protein targeting systems for these bacteria. These developments are important in the food industry where lactic acid bacteria can be exploited as food-grade cell factories.

Gene Expression Regulation, Bacterial↗

PubMed2026

Model-driven analysis reveals oxidative stress adaptation enabling efficient energy utilization in a Crabtree-negative Saccharomyces cerevisiae.

Although abolishing the Crabtree effect in Saccharomyces cerevisiae through a pyruvate dehydrogenase bypass eliminates carbon loss through ethanol overflow metabolism, it compromises growth rates. While the Crabtree effect has been a valuable natural adaptation, it is energetically inferior to respiration and is generally undesirable in cell factories engineered to produce assimilatory compounds. Restoring growth efficiency in Crabtree-negative strains remains a central challenge. Through adaptive laboratory evolution of the engineered strain (sZJD23) and subsequent reverse engineering, a variant (sZJD28) with markedly improved growth was identified. This improvement is driven primarily by a mutation in MED2 (encoding a Mediator complex subunit) and, to a lesser extent, a mutation in GPD1 (encoding glycerol-3-phosphate dehydrogenase). By integrating quantitative proteomics with enzyme-constrained genome-scale modelling, we demonstrate that these mutations jointly enable a more efficient mode of oxidative stress adaptation and energy utilization. The GPD1 mutation suppresses a protein-costly, suboptimal NAD⁺-recycling strategy reliant on glycerol synthesis, while the MED2 mutation reshapes the oxidative stress response towards peroxisomal detoxification. Collectively, these adjustments optimize metabolic flux distribution and reduce protein costs in energy metabolism, thereby increasing ATP availability. Our findings reveal how coordinated mutations in regulatory and metabolic genes restore growth fitness in engineered Crabtree-negative yeast.

Saccharomyces cerevisiae↗

PubMed2024

Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Saccharomyces cerevisiae↗

PubMed2024

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Photosynthesis↗

PubMed2006

Genomics and blood substitutes for 21st century Europe ("EuroBloodSubstitutes").

Blood transfusion is a medical intervention practised throughout the world. Blood is a biologically active material that can transmit diseases (e.g., HIV/AIDS and, perhaps, vCJD). People are becoming increasingly concerned about blood safety, despite improved screening and processing. Consequently, they are reluctant to donate blood or receive transfusions. Such problems can be solved by the development and incorporation into transfusion practices of so-called "blood substitutes" to replace some blood uses. The EuroBloodSubstitutes Project is funded by the European Union Framework 6 Programme to develop a technological platform for producing novel haem proteins and blood substitute components using micro-organisms (bacteria, fungi, yeast) as "cell factories." The Project will focus on bacteria (Escherichia coli), yeast (Pichia pastoris) and, longer-term, filamentous fungi (Aspergillus niger), all organisms used to synthesize commercially important products. The multi-centre Consortium consists of the Universities of (1) Nottingham (UK), (2) Essex (UK), (3) Denmark Technical (Denmark), (4) Lund (Sweden), (5) Milan (Italy), (6) Nancy (France), (7) Parma (Italy), (8) Rome "La Sapienza" (Italy), (9) Semmelweis (Hungary), together with (10) Alligator Bioscience, AB (Sweden), (11) LCC Engineering & Trading GmbH (Switzerland), (12) Scottish National Blood Transfusion Service (UK), and (13) Sanquin Bloodbank (The Netherlands). The EuroBloodSubstitutes Project will be informed by lay and professional stakeholders (e.g., clinicians, blood donors, patient groups, prescribers and policy makers). Outcomes of the Project are (1) the production of an information pack, decision aids and physician training aids, giving balanced overviews of the benefits and risks of transfusion of blood or potential substitutes, and (2) an interactive web site (http//:www.eurobloodsubstitutes.com) for information dissemination. This will improve knowledge and address misunderstandings about transfusion issues in a climate of changing patient expectations on blood safety and benefits of blood substitutes.

Biotechnology↗

PubMed2025

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing.

The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Xylose↗

PubMed2025

Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.

A large variety of synthetic biology toolkits for the introduction of multiple expression cassettes is available for Saccharomyces cerevisiae. Unfortunately, none of these tools is designed to allow the modification - exchange or removal - of the cassettes already integrated into the genome in a standardized way. The application of the modularity principle therefore ends to the steps preceding the final host engineering, making microbial cell factories construction stiff and strictly sequential. In this work, we describe a system that easily allows CRISPR-mediated swapping or removal of previously integrated cassettes, thus bringing the modularity to the strain level, enhancing the possibility of modifying existing strains with a reduced number of steps. In the system, each cassette is tagged with specific barcodes, which can be used as targets for CRISPR nucleases (Cas9 and Cas12a), allowing the excision of the construct from the genome and its substitution with another expression cassette or the restoration of the wild type locus in one single standardized step. The system has been applied to the previously developed Easy-MISE toolkit and tested by swapping fluorescent protein expression cassettes with an efficiency of ∼90% quantified by PCR and flow cytometry.

Saccharomyces cerevisiae↗

PubMed2026

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass.

Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis-degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.

Talaromyces↗

PubMed2003

Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Bacillus Phages↗

PubMed2000

The alpha-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization.

Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425. Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose. The type II mutant was affected in the gene responsible for the synthesis of the extracellular alpha-amylase. This gene (amyH) was isolated by functional complementation of mutant II and sequenced. The deduced protein (AmyH) showed a high degree of homology to a proposed family of alpha-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals. AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids. The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli. AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential. In addition, H. meridiana and H. elongata were able to secrete the thermostable alpha-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes.

Amino Acid Sequence↗

PubMed1992

Production and characterization of recombinant mouse neurotrophin-3.

Neurotrophin-3 (NT-3) is a neurotrophic-factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 micrograms purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.

Amino Acids↗

PubMed2004

The Bacillus secretion stress response is an indicator for alpha-amylase production levels.

AIMS: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high-level alpha-amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production. METHODS AND RESULTS: The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B. subtilis strains. TepA was shown to be dispensable for secretion stress management. Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production. CONCLUSION: Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels. SIGNIFICANCE AND IMPACT OF STUDY: Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory.

Acyltransferases↗