1,721,044 research outputs found
Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii
No full textIn order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 microns sequence yield stable transformants. We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids
Selfish and cooperative strategies, two energetic behaviors driven by environmental constraints
No full textNutrient richness, and specifically the abundance of mono- and disaccharides that characterize several food matrixes, such as milk and grape juice, has allowed the speciation of lactic acid bacteria and yeasts with a high fermentation capacity instead of energetically favorable respiratory metabolism. In these environmental contexts, rapid sugar consumption and lactic acid or ethanol production, accumulation and tolerance, together with the ability to propagate in the absence of oxygen, are several of the ‘winning’ traits that have apparently evolved and become specialized to perfection in these fermenting microorganisms. Here, we summarize and discuss the evolutionary context that has driven energetic metabolism in food-associated microorganisms, using the dairy species Lactococcus lactis and Streptococcus thermophilus among prokaryotes and the bakers’ yeast Saccharomyces cerevisiae among eukaryotes as model organisms
Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast
No full textMethod of flow cytometric analysis have recently been developed that make it possible to obtain segregated data on a single cell basis. In particular, it has been previously demonstrated that protein distributions obtained by flow cytometry give information about the law of growth of the cell population and the law of growth of the single cell; thus these distribution show how the microbial population is actually growing at the moment of the analysis and may yield more accurate and predictive information. We have extended the analysis of protein distribution and cell volume distribution to continuous cultures of Saccharomyces cerevisiae growing in a glucose-limited chemostat. We have found that: (1) to each dilution rate corresponds a given protein and volume distribution that does not change with time in steady state cultures; (2) there is a good proportionality between the average cell volume and the average protein content; (3) the protein distribution obtained can be easily analyzed with the model of growth of yeast previously developed in our laboratory; (4) the analysis of perturbed states shows that both protein distribution and volume distribution change very quickly; thus they are very sensitive parameters and can be used for monitoring and controlling industrial fermentation
Glycerol production in a triose phosphate isomerase deficient mutant of Saccharomyces cerevisiae
No full textInteresting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemicals. In this paper, we show that engineered yeast cells deficient in the triose phosphate isomerase activity are able to produce glycerol without the use of steering agents. High yields of conversion of glucose into glycerol (80-90% of the theoretical yield) and productivity (1.5 g L(-1) h(-1)) have been obtained by a bioconversion process carried out in a poor and clean medium. We obtained indications that the growth phase at which the biomass was collected affect the process. The best results were obtained using cells collected at the end of exponential phase of growth. In perspective, the strategies and the information about the physiology of the cells described here could be useful for the developing of new biotechnological processes for glycerol production, outflanking the problems related to the use of high level of steering agents
Factors affecting glycerol production by a bioconversion process with a triose phosphate isomerase deficient mutant of Saccharomyces cerevisiae
No full textThe economics of bioglycerol production depend on several factors such as selection of suitable microorganism, exploitation of inexpensive raw materials and optimization of the process. In this work we studied the influence of some parameters on the bioconversion process for the production of glycerol from glucose using a S. cerevisiae strain lacking triose phosphate isomerase activity. The results indicate that the mode of addition of carbon source affects glycerol yield. The bioconversion process can be carried out in a medium containing only glucose and phosphate; a glucose concentration over 20 g/L must be maintained in order to obtain high yield and productivity. The best growth condition to obtain efficient biomass for the bioconversion process is on rich media containing 1.5% ethanol and 0.1% glucose. It is important to collect the biomass at the end of the exponential phase of growth and after an induction in the presence of 0.1% glucose
Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system
No full textAutoselection systems allow the selection of a genetically engineered population independently of the growth medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed. By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level. Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the cellular plasmid distribution on the dynamics of growth are also discussed
PRODUCTION OF FRUCTOSE DIPHOSPHATE BY BIOCONVERSION OF MOLASSES WITH SACCHAROMYCES-CEREVISIAE CELLS
No full textSugar beet molasses was used as carbon source for Saccharomyces cerevisiae growth and as substrate for bioconversion to fructose diphosphate. The highest level of fructose diphosphate (26.6 g/L) was reached after 10 h incubation of permeabilized cells under appropiate molasses and phosphate to cell ratio and represented a 64% yield of bioconversion
COPY NUMBER MODULATION IN AN AUTOSELECTION SYSTEM FOR STABLE PLASMID MAINTENANCE IN SACCHAROMYCES-CEREVISIAE
No full textEfficient expression of a foreign gene requires a stable vector present at a high number of copies per cell. We have constructed an autoselection system for the stable maintenance of expression vector in the yeast Saccharomyces cerevisiae that uses the fructose 1,6-bisphosphate aldolase gene (FBA1) to stabilize plasmids in cells bearing a disruption of the chromosomal FBA1 gene. This system allowed us to obtain stable production of a reporter heterologous enzyme (Escherichia coli beta-galactosidase) in rich media. By using an inducible promoter to regulate the expression of FBA1 gene, we have also obtained the modulation of plasmid copy number by carbon source
How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis
No full textThe optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels
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