Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant.

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media.

Current awareness.

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals - search completed 7th Mar. 2001)

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells.

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts.

Interest in the production of L-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovine L-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into a K. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter(-1), 0.91 g liter(-1) h(-1), and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those usual for bacterial processes.

Isolation, nucleotide sequence, and physiological relevance of the gene encoding triose phosphate isomerase from Kluyveromyces lactis.

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae DeltaTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Control by nutrients of growth and cell cycle progression in budding yeast, analyzed by double-tag flow cytometry.

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual Saccharomyces cerevisiae cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing S. cerevisiae populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.

Identification of different daughter and parent subpopulations in an asynchronously growing Saccharomyces cerevisiae population.

Under all growth conditions, a growing Saccharomyces cerevisiae yeast population is extremely heterogeneous, since individual cells differ in their cell size; this is due to their position in the cell division cycle and their genealogical age. To gain insight into the structure of a growing yeast population, we used a recently developed flow cytometric approach which enables, in asynchronously growing S. cerevisiae populations, tagging of both the cell age and the protein content of individual cells. This approach enabled the identification of daughter cells belonging to different cell cycle positions (i.e. newborn, G1, S + G2 + M + G1*, and dividing), thus yielding information about the relative fraction in the whole population, cell size and variability. More limited information could be obtained for the parent subpopulation; however, we were able to identify and characterize the dividing parent cells. The coefficient of variation (CV) of the protein content distribution for the dividing parents (27) was much higher than the CV of dividing daughters (18). Further findings obtained indicated a large overlap between the cell protein content distributions of daughter and parent cells as well as between the protein content of cells of the same subpopulation but belonging to different stages of the cell division cycle. The analysis of these differences enables a better understanding of the complex structure of an asynchronously growing yeast population.

Glycerol production in a triose phosphate isomerase deficient mutant of Saccharomyces cerevisiae.

Interesting 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.

Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system.

Autoselection 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.

Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells.

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspectives are discussed.

A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth.

Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell. In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations.

Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid.

Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemical(s). In this paper, we describe the accumulation of lactic acid in the culture medium of growing, engineered yeast cells expressing a mammalian lactate dehydrogenase gene (LDH-A). High and reproducible productions (20 g/L) and productivities (up to 11 g/L/h) of lactic acid have been obtained by modulating the physiological growth conditions. Since yeast cells are acid tolerant and survive at very low pH values, the production of lactate can be avoided. In perspective, the approaches described could be useful for the production of lactic acid, outflanking the problems related to the synthesis from bacteria cells. In fact, during industrial productions, there is an inhibitory effect on the metabolic activities of the growing bacteria (i.e., Lactobacillus spp.) caused by the acid produced and by the low pH value. Thus, strategies to prevent the lowering of pH are conventional operations. These processes allow the production of lactate(s) and require the purification of the acid from its salt. The biotechnological implications of this study are also discussed.

Copy number modulation in an autoselection system for stable plasmid maintenance in Saccharomyces cerevisiae.

Efficient 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.

Bioconversion of lactose/whey to fructose diphosphate with recombinant saccharomyces cerevisiae cells.

Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli beta-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. We showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

Alteration of cell population structure due to cell lysis in Saccharomyces cerevisiae cells overexpressing the GAL4 gene.

Transformed Saccharomyces cerevisiae cells overexpressing the Escherichia coli LacZ gene and the transcriptional activator GAL4, release in the external medium a fraction (from 2 to 10%) of the total beta-galactosidase activity (Porro et al., 1992b). It is known that this abnormal release of a cytoplasmic protein is related to a partial cell lysis of the yeast population, which is likely to be caused by the overexpression of the transcriptional activator GAL4. In the present paper we have characterized the GAL4-induced cell lysis phenomenon. The expression of the GAL4 gene causes morphological modifications and alteration of the cell size distribution. The cell lysis is independent of the expression of the heterologous LacZ gene and occurs in a specific subpopulation of cells (the parent cells) independently of the genealogical age, growth phase conditions and cell cycle progression. Lysis is preceded by a loss of the plasma membrane integrity as indicated by the uptake of ethidium bromide in unfixed cells. Computer analysis of simulated protein distributions indicates that cell lysis takes place in a sizeable aliquot (about 50%) of the parent cells, therefore profoundly altering the age structure of the population.

In Saccharomyces cerevisiae, protein secretion into the growth medium depends on environmental factors.

In the budding yeast Saccharomyces cerevisiae the cell wall, mainly composed of mannoproteins and glucans, constitutes a barrier to protein excretion in the growth medium. In this paper we have studied the effects of different environmental parameters on excretion of Escherichia coli beta-galactosidase obtained by exploiting the glucoamylase II signal sequence. Excretion of the unglycosylated beta-galactosidase was detectable only in cells grown in rich medium, was affected by temperature (36 degrees C > 30 degrees C >> 24 degrees C) and slightly stimulated by reducing agents. On the contrary, glycosylated proteins, such as alpha-galactosidase and glucoamylase II, were excreted to a good extent under all tested conditions of medium composition, growth temperature and pH. These data indicate that optimization of environmental parameters may help the excretion of heterologous proteins, offering advantages for protein purification.

Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells.

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.

Enhanced expression of heterologous proteins by the use of a superinducible vector in budding yeast.

We report the effects of a strong overexpression of the GAL4 activator protein on the expression of UASGAL regulated genes, obtained by cloning the GAL4 gene and the GAL1-10 upstream activating sequence (UASGAL)-lacZ fusion in the same high copy number plasmid. Comparable amounts of active enzyme were obtained by host strains usually producing different levels of cloned proteins due to their different genetic background. The transformed cells constitutively produced low levels of beta-galactosidase (1-2% of total proteins) both in glucose and in raffinose minimal media. Nevertheless, expression was still inducible and a tenfold induction could be rapidly obtained by the addition of 0.5% (w/v) galactose to the culture, even when glucose was still present in the medium.