65 research outputs found
The incorporation of mannoproteins in the cell wall of S. cerevisiae and filamentous Ascomycetes
Науково-документальна серія книг «Реабілітовані історією»: досвід та перспективи наукового дослідження (до 95-річчя з дня народження академіка НАН України П. Т. Тронька)
У статті аналізується наукова та громадсько-політична діяльність академіка НАН України П. Т. Тронька як науковця та організатора реалізації Державної програми науково-документальної серії книг «Реабілітовані історією».В статье анализируется научная и общественно-политическая деятельность академика НАН Украины П. Т. Тронько как учёного и организатора реализации Государственной программы научно-документальной серии книг «Реабилитированные историей».The author analyzed research, political and public activity of academic P. Tronko as scholar and organizer of State program of documentary series «Rehabilitated by history»
Microbiological and biochemical aspects of the fermentation of milk with saccharomycopsis lipolytica
Applied Science
Correlations between sequences and production (AGGREGATION CH 2)
A set of 70 variable domains of heavy chain Camelid antibodies (VHHs) were grown under identical conditions in yeast. The production yields in Saccharomyces cerevisiae ranged between nearly 0 and 150 mg/l. To understand these large differences in production of VHHs, even those recognizing the same antigen or those with a high sequence similarity, we searched for correlations between a series of aspects of the sequences and features of 3D structures and the production yield. Several parameters on DNA sequence and protein sequence level were analysed using simple bioinformatics techniques. No trivial correlations were found. We did notice many non-optimal codons. Visual inspection of the multiple sequence alignment revealed an enrichment of certain amino acid types at certain positions in the poorly produced VHHs. These findings resulted in the studies described in more detail in chapter 5
Correlations between sequences and production (THESIS VERSION)
A set of 70 variable domains of heavy chain Camelid antibodies (VHHs) were grown under identical conditions in yeast. The production yields in Saccharomyces cerevisiae ranged between nearly 0 and 150 mg/l. To understand these large differences in production of VHHs, even those recognizing the same antigen or those with a high sequence similarity, we searched for correlations between a series of aspects of the sequences and features of 3D structures and the production yield. Several parameters on DNA sequence and protein sequence level were analysed using simple bioinformatics techniques. No trivial correlations were found. We did notice many non-optimal codons. Visual inspection of the multiple sequence alignment revealed an enrichment of certain amino acid types at certain positions in the poorly produced VHHs. These findings resulted in the studies described in more detail in chapter 5
Dynamic optimal metabolic control theory: a cybernetic approach for modelling of the central nitrogen metabolism of S. cerevisiae
The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest. In the DOMC approach the cell is regarded as an optimally controlled system. Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment. The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation. The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data. It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways. The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant. The simulation results are validated with experimental data
The role of ammonia metabolism for nitrogen catabolite repression in Saccharomyces cerevisiae
Saccharomyces cerevisiae is able to use a wide variety of nitrogen sources for growth. Not all nitrogen sources support growth equally well. In order to select the best out of a large diversity of available nitrogen sources, the yeast has developed molecular mechanisms. These mechanisms consist of a sensing mechanism and a regulatory mechanism which includes induction of needed systems, and repression of systems that are not beneficial. The first step in use of most nitrogen sources is its uptake via more or less specific permeases. Hence the first level of regulation is encountered at this level. The next step is the degradation of the nitrogen source to useful building blocks via the nitrogen metabolic pathways. These pathways can be divided into routes that lead to the degradation of the nitrogen source to ammonia and glutamate, and routes that lead to the synthesis of nitrogen containing compounds in which glutamate and glutamine are used as nitrogen donor. Glutamine is synthesized out of ammonia and glutamate. The expression of the specific degradation routes is also regulated depending on the availability of a particular nitrogen source. Ammonia plays a central role as intermediate between degradative and biosynthetic pathways. It not only functions as a metabolite in metabolic reactions but is also involved in regulation of metabolic pathways at several levels. This review describes the central role of ammonia in nitrogen metabolism. This role is illustrated at the level of enzyme activity, translation and transcriptio
Llama heavy chain antibodies, to mutate or not to mutate (AGGREGATION CH 4)
A high production yield and good stability are required for the large scale application of llama heavy chain antibody fragments (VHHs) against targets such as a microbicide against HIV. Many aspects of the production process are amenable to optimization by engineering. Codon usage, for example, can be improved, but it is also possible to remove proteolytic cleavage sites, chaperone binding sites, or to improve the packing of the protein by increasing the numbers of hydrophobic contacts or hydrogen bonds. However, not every amino acid can be mutated freely. Obviously, the cysteine bridge should stay intact at all cost. It also seems unwise to mutate residues involved in scaffolding the CDRs. We have made a large series of in silico studies, and tabulated and analyzed all known mutations in VHHs. No mutation information was available for eight residues that were predicted to be important for folding and production yield using the rules for in vitro folding of Vendruscolo. When we mutated these eight residues we observed thermostability differences ranging from -1 to -14 degrees in agreement with what one would expect from the selected modifications. The production yield of most of the mutants decreased as well. All known and predicted mutation effects were combined in a generic 'VHH-mutability' table. This table could be used to flawlessly predict the best producing variants when a series of HIV neutralizing VHH variants was obtained
The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in the central nitrogen metabolism
Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source. GOGAT's importance for cellular metabolism is still unclear. For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (¿glt1) mutant strain (VWk274 LEU+) was studied in glutamine-limited continuous cultures. As reference, we did the same experiments with a wild-type strain (VWk43). Intracellular and extracellular metabolites were analysed during different steady states in both strains. The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively). The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria
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