The promoter of Saccharomyces cerevisiae FBA1 gene contains a single positive upstream regulatory element.

The glycolytic enzyme fructose 1,6-bisphosphate aldolase is encoded by the FBA1 gene of Saccharomyces cerevisiae. Transcription of aldolase gene is not regulated by glucose and high levels of expression have been observed also during growth on nonfermentable carbon source. A FBA1::lacZ gene fusion was constructed and a deletion analysis demonstrated the presence of a unique cis-acting positive upstream element (UAS) required for high levels of FBA1 expression. This element is located between positions -550 and -440 upstream of the aldolase open reading frame and it contains sequences known to constitute the binding sites for the multifunctional proteins RAP1 and ABFI and two TTCC motifs.

Development of a pH-controlled fed-batch system for budding yeast.

In the manufacturing of baker's yeast by aerobic fed-batch systems, continuous assessment of the state of the process is necessary for regulating the flow rate (on/off) for growth medium addition. A new, simple method for the fed-batch yeast process has been developed. It is based on pH changes as a suitable parameter for regulating the feed of fresh concentrated medium in response to metabolic activities of the yeast population. Experimental results have shown that it enables the attaining of high cell density with both high productivity and high yields.

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.

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.

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.

Flow cytometry and cell cycle kinetics in continuous and fed-batch fermentations of budding yeast.

Cell size distributions, obtained either as protein distribution by flow cytometry or as cell volume distribution by a Coulter counter, give relevant information about the growth conditions of populations of budding yeast Saccharomyces cerevisiae. We have previously found a good correlation between these distributions and the growth rate in continuous cultures (Ranzi et al., Biotechnol. Bioeng. 1986, 28, 185-190). We now present determinations of the protein distributions and cell volume distributions during different fed-batch fermentations performed with a simple on/off controller. Since during the fed-batch fermentation a true steady state is not obtained, the distributions continuously change with time, but nevertheless we observed a good correlation between the average of both distributions and the actual growth rate. The behavior of the cell size distributions can be interpreted on the basis of a two-threshold cell cycle model in which both the critical protein content at budding (Ps) and the critical protein content for cell division (Pm) are differently modulated by the growth rate. Additional findings will be presented showing that this model can be used to successfully explain the insurgence and the maintenance of oscillatory states in continuous cultures.

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.

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.

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast.

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.

Heterologous gene expression in continuous cultures of budding yeast.

In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements.

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.

Efficient production of recombinant DNA proteins in Saccharomyces cerevisiae by controlled high-cell-density fermentation.

High levels of expression of heterologous proteins (from 5 to 15% of total cell proteins) in the budding yeast Saccharomyces cerevisiae have been obtained previously by the use of the inducible strong hybrid promoter UASGAL/CYC1, in batch as well in continuous cultures. However, in order to maximize the yield of heterologous proteins, a computer controlled fed-batch fermentation is essential. For this reason we have developed a fed-batch system based on a semiconductor gas detector that measures ethanol in the outflow gases. The optimal conditions are described for very high production (up to 1550 mg/liter), with both high productivity (up to 100-120 mg/liter/h) and high yield (up to 15 mg of protein/g of dry biomass), of heterologous protein driven by the UASGAL/CYC1 promoter in a completely computer controlled fed-batch fermentation of budding yeast. However, high production was dependent upon the addition of a large amount of galactose. The process was improved by developing a new, more easily inducible, vector system obtained by subcloning the GAL4 gene.

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.

Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii.

In 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.

Oscillations in continuous cultures of budding yeast: a segregated parameter analysis.

Sustained oscillations have been observed in continuous cultures of Saccharomyces cerevisiae. These oscillations appear spontaneously under aerobic conditions and may constitute a severe limitation for process control. We have found that oscillations arise only in a well defined range of dilution rates and dissolved oxygen values. The period of the oscillations is related, but not equal, to the mass doubling time, and shows a relation ship with both the parent cells and daughter cells generation times. At high dilution rates two oscillatory regimens, with different periods, are observed. The analysis of the budding index shows a marked degree of synchronization of the culture, however significant differences, both in phase and in amplitude, are ob served if the budding index of parent cells and of daughter cells are considered separately. The complex changes of the cell population are clearly demonstrated by the continuous and periodic modification of both cell volume distributions and protein distributions. Ethanol is always accumulated before the drop of dissolved oxygen concentration and one of the peaks of budding index. We propose a model that explains the insurgence of these oscillation as a consequence of changes in cell cycle parameters due to alternate growth in glucose and in ethanol.

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.

Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast.

Method 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.

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.