Traceability is crucial in modern food technology, and it is thus essential to develop a method to discriminate between different
In the present chapter, we first review the current knowledge regarding the application of
Torulaspora delbrueckii in wine production
Saccharomyces cerevisiae and non- Saccharomyces species in wine making: A brief overview
Flavours and aromas of wines are due to the grape itself and to biological activities carried out by the microorganisms. Several species of yeasts and bacteria and sometimes filamentous fungi may be present during fermentation of the must, and are responsible for the final characteristics of the wine . Besides
2.2. The use of
Torulaspora delbrueckii in the wine industry
The use of non-
Torulaspora delbrueckii in bread production
3.1. Baker´s yeast and its important traits for baking applications
In the history of human nutrition, a diversity of bakery products has been created which continues today. Bread is mostly made from flour dough that is allowed to rise (leaven) before baking in the oven. Making bread requires three main ingredients: flour, water, and yeast. The yeast’s main role in the bread making process is to promptly ferment the sugars available in the flour of the dough or that have been added to it. As a result of an efficient fermentation, the yeast produces carbon dioxide (CO2] and ethanol; the CO2 is trapped within the gluten matrix of the dough, causing the leavening or rising, while the ethanol contributes to flavour development, along with other volatile compounds and flavour precursors that are formed during the fermentation process. Technically, the most important properties of baker’s yeast comprise  the leavening ability in the dough,  the ability to adapt to different carbon sources, by expressing invertase and maltase activities, and  stress resistance, particularly osmo- and cryo-tolerance. Obviously yeast should also contribute to the flavour of the baked products, as well as grow rapidly in molasses, which are used in the culture media in their industrial production. Commercial baker's yeasts are domesticated strains, essentially of
The physiological requirements of baker’s yeast for optimal production and application represent an apparent contradiction (Figure 1]. In fact, sugar-limited respiro-fermentative fed batch cultivation (yeast production phase) must render a yeast product that has developed a high fermentative capacity, although this requirement is not important during this phase. Subsequently, the gassing capacity (fermentation) is used in the application phase in the dough, under anaerobic, excess sugar conditions. Therefore the physiological flexibility of baker's yeast must be exceptional.
In addition to good fermentative capacities and high stress resistance, another major trait must be considered when selecting a yeast strain for the baking industry : effective biomass production in molasses. Because molasses are cheap and easily available and contain some nitrogen and several vitamins and minerals necessary for yeast growth, they are the main substrate used for large-scale baker’s yeast production. However, molasses are considered a major factor of variation in the quality of baker’s yeast . These substrates are highly variable and contain different proportions of sugars. Though sucrose is the major sugar present, there is also a quite high amount of glucose and fructose. Sucrose is cleaved outside the cell by invertase into glucose and fructose. Invertase is also capable of cleaving raffinose, a trisaccharide also present in molasses, into fructose and melibiose (glucose-galactose), but melibiose is generally not assimilated . The main fermentable sugar in plain bread dough is maltose, liberated from starch by amylase activity (α-glucosidase) in flour. This disaccharide is transported through a maltose permease and is subsequently hydrolyzed into glucose by maltase (figure 2]. The order by which these different carbohydrates are fermented by
For the most part, regulation is mediated by catabolite repression, acting at early steps in various catabolic pathways. The aim of the regulation is to induce utilization of most favoured carbon source (glucose), and to exclude utilization of other carbon sources if a sufficient amount of glucose is available. Therefore, in most strains of
Torulaspora delbrueckii as an emergent yeast in the baking industry
Nowadays, the baker’s yeast strains used have been developed as a result of centuries of experience and selection, resulting in a high degree of domestication best suited for bread making. Nevertheless, research to improve yeast strains continues. Although methods of classical genetics (selection, mutation, and hybridization) are still very useful, novel methods such as protoplast fusion and genetic engineering have resulted in baker’s yeast strains with even better technological properties [35, 37, 44].
Although this yeast is widely commercialized in Japan, the regular utilization of this species in the bread-making industry has not been established due to several drawbacks.
4. Sugar metabolism in
As referred above,
4.1. Sugar utilization patterns and respiro-fermentative metabolism
One of the leading characteristics of a valuable baker’s yeast is its dough-leavening ability, which implies there is efficient fermentation of both the maltose and glucose that are present in the dough. Another important feature is its ability to generate high biomass yields on sucrose, the major sugar present in molasses. On the other hand, glucose and fructose are the main sugars present in grape musts, and thus efficient fermentation of these sugars is also of great importance for utilization of a yeast strain in wine fermentation
The behavior of
Aiming to provide new insights into the molecular mechanisms underlying energy source signaling in
In summary, the overall patterns of sugar utilization and regulation in mixed sugar media by
4.2. Hexose transport in
The sugar porter family is the largest within the major facilitator superfamily (MFS), which includes proteins from Bacteria, Achaea, and Eukarya, with very diverse sequences and function [55-57]. Proteins belonging to the MFS exhibit highly structural conservation, though they share little sequence similarity . Generally, these permeases have 12 putative transmembrane segments, and consist of a single integral membrane protein with two sets of six hydrophobic transmembrane-spanning (TMS) (-helices connected by a hydrophilic loop, whose amino- and carboxy-terminal regions localize to the cytoplasm [59-61]. The strong similarity between the two sets of hydrophobic TMS of MFS proteins and their overall structure supports the theory that they result from a gene duplication event that probably took place before de divergence of MFS families [57, 59]. Sugar transport across the plasma membrane is the first and obligatory step of its utilization. Yeasts can use different carbon sources for growth, but evolution has selected mechanisms for the preferential utilization of glucose. As permeability of biological membranes is quite restricted, most of the cellular nutrients must enter the cell via specific transport systems and both facilitated diffusion and proton-symport transport systems for sugars have been described in yeasts. In facilitated diffusion, solutes are transported down a concentration gradient by a uniport mechanism. Secondary active transport uses accumulated energy from an electrochemical gradient to transport molecules against their concentration gradient, coupled with the simultaneous movement of another molecule (normally H+ or Na+) in the same (symport) or opposite (antiport) direction . Such a mechanism becomes fundamental during growth in very low extracellular sugar concentrations, when an intracellular accumulation of hexoses may be necessary to allow hexose kinases to function optimally. Evidently, yeast species possessing proton-hexose symport systems are better adapted to grow in low hexose concentrations [63, 64]. Since a facilitated diffusion transport system is most efficient only under reasonably constant levels of the carrier substrate, this system might not be appropriate for yeasts like
Multiple hexose carriers have been characterized genetically in
The number of hexose transporters is very variable among yeasts, ranging from 20 hexose transporters in
Two natural habitats of Torulaspora delbrueckii are bread doughs and fruit juices, such as grape juice, environments that are rich in sugars. As a consequence of growth and fermentation of these sugars, this yeast experiences dramatic changes in its physicochemical environment, and thus must adapt to these varying conditions in order to sustain its growth. The sugar concentration may decline from 1 M to 10–5 M during fermentation, and the overall composition of the medium will be altered by yeast metabolism. The sugar transport activity of the cell and the proteins that mediate sugar transport must be responsive to these changing conditions, and thus the capacity and kinetic complexity of hexose transport in the yeast may be a reflection of the existence of a large number of sugar transporter genes in its genome. Based on this assumption, multiple hexose transporters with different affinities for glucose probably exist in T. delbrueckii. This yeast displays a mediated glucose transport activity best fitted assuming a biphasic Michaelis–Menten kinetics with a low- (apparent Km = 8.32 ± 0.55 mM) and a high-affinity component (apparent Km = 1.30 ± 0.34 mM) . A kinetic compatible with the presence of these two components was observed in either glucose-, frutose- or maltose-grown cells. Aiming to identify glucose transporters in T. delbrueckii, a complementation screen of a S. cerevisiae hexose transport-null mutant strain  yielded a genomic DNA fragment containing a gene encoding Lgt1p, a low-affinity glucose transporter . When expressed in the hxt null strain, Lgt1p exhibited an apparent Km value for glucose of 36.5 ± 3.1 mM, in the range of the low-affinity component, and a Vmax of 1.1 ± 0.04 nmol/s/mg dry weight. This transporter is also able to mediate significant fructose uptake in the hxt mutant, although with a lower affinity than that for glucose, with an apparent Km value of 51.4 ± 3.0 mM. Glucose transport in this mutant (expressing Lgt1p) was inhibited by the presence of frutose, manose and maltose. Expression studies of the T. delbrueckii LGT1 gene in S. cerevisiae strains, including wild-type, using a fusion of the LGT1 promoter to the reporter gene lacZ, revealed that it was induced by high glucose concentrations, and its expression was elevated in media containing 4% glucose and almost undetectable in medium containing galactose as the sole carbon source. The transcription factor Rgt1p was necessary for repression of LGT1 in the absence of glucose; however, and in contrast with the activity of Rgt1p as a bifunctional regulator in S. cerevisiae strains, full induction of LGT1 by high glucose concentrations does not require functional Rgt1p. Even though Mig1p-binding sequences were identified in the promoter region of the LGT1 gene, the general repressor of S. cerevisiae had no effect in the regulation of LGT1 gene expression. However, disruption of MIG2 in a mig1 background led to high levels of LGT1 expression in high glucose concentrations, indicating that either Mig2p or both Mig1p and Mig2p acting redundantly, function as repressors of LGT1 expression under these conditions, consistently with their function in S. cerevisiae.
Even though just one glucose transporter has been identified in T. delbrueckii until now (the low-affinity glucose transporter LGT1 ), it is likely that T. delbrueckii possesses high-affinity transporters, which is supported by the biphasic Michaelis–Menten kinetics of glucose transport. These results suggest there are additional physiological relevant glucose transporters. Identification of novel transporters will provide new clues regarding the mechanisms underlying regulation of sugar transport, and as a consequence the fermentative capacity of this biotechnologically relevant yeast.
4.3. Cloning and characterization of the Torulaspora delbrueckii MAL11, encoding a high-affinity maltose transporter
The genes involved in the utilization of maltose have been characterized in detail in laboratory strains of Saccharomyces cerevisiae . Genetic analysis revealed there are five MAL loci, MAL1-MAL4 and MAL6, located on different chromosomes , but with a high degree of similarity . Each locus contains a set of three different genes that encode a maltose transporter (MALT - MALx1, where x represents the number of each locus), α-glucosidase (MALS - MALx2], and a regulatory protein (MALR - MALx3] . MALx1 genes code for high affinity maltose-H+ simporters with a Km of approximately 5 mM , but with different specificities for various substrates. While Mal11p/Agt1p transports a wide range of substrates, including several α-glucosides , Mal31p and Mal61p seem to primarily use maltose, maltotriose, and turanose [89, 90]. Nevertheless, only one fully functional locus seems to be found in standard laboratory strains, MAL1, which is heavily regulated through repression by glucose and induction by maltose . Comparatively, industrial yeasts contain multiple fully or partially functional MAL loci [92, 93]. Additional analysis showed there are considerable variations in the MALR gene , leading to non-sensitivity to glucose and lack of control by maltose. These special features were the result of applying successive programs directed to a rapid adaptation to the fermentation of maltose.
Characterization of maltose transport rates in Torulaspora delbrueckii indicated it contains an inducible active transport system that co-transports protons with maltose, with the following kinetic parameters: Vmax 1.03 ± of 0.05 nmol s-1 (mg dry weight) -1 and Km 2.26 ± 0.27 mM maltose . This transport system was subject to glucose repression and was competitively inhibited by the presence of sucrose, melizitose, and melibiose, suggesting these sugars likely share the same transporter(s) with maltose.
A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11] was isolated by complementation cloning in S. cerevisiae with a T. delbrueckii genomic library [95, 96]. DNA sequence analysis revealed the presence of an ORF of 1884 bp encoding a putative 627-amino acid membrane protein with 10 transmembrane domains, highly similar to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert included a partial ORF (TdMAL12] on the opposite strand and direction, highly similar to the S. cerevisiae MAL12 gene. Two consensus binding sites of the repressor Mig1p (positions -362 to -378 and -459 to -475] are evident in the divergent promoter. Interestingly, the first overlaps with a binding site of a MAL activator (-451 to -461]. A similar situation was described in the intergenic region MAL61-MAL62 in S. cerevisiae, where one of the two Mig1p binding sites is very close to a UASMAL site . This overlap seems to have a functional role in the transcriptional regulation of MAL61 and MAL62 genes, as occupation of the UASMAL site will result from direct competition between the two regulators for the binding region . Sequence analysis, Northern blot, and transport measurements, indicated that TdMAL11 expression is regulated by carbon source, and is subjected to repression by glucose and induction by maltose. Attempts to disrupt TdMAL11 and Southern blot analyses revealed the presence of two functional MAL loci. Disruption of a single copy decreased the Vmax of maltose transport, but not the Km, whereas the double disruption abolished the uptake of this sugar in T. delbrueckii .
As referred above, the activity and regulation of the maltose uptake system in T. delbrueckii cells could be a good target for improvement of its leavening ability in bread dough, and the identification of maltose transporter genes in this yeast can now open the door to these studies.
5. Freezing tolerance of Torulaspora delbrueckii: Cellular and biochemical basis
In the baking industry, yeasts encounter numerous stresses. During production, they must adapt to low sugar and high aeration, repressing fermentation to produce large amounts of biomass. Cells are then preserved in a cold, frozen or dry state until use, when rehydration or thawing and inoculation cause osmotic shock in a new environment that requires the induction of enzymes for maltose utilization under semianaerobic conditions. The low stress resistance of yeast during active fermentation is disadvantageous for its use in industrial applications, and it would be highly advantageous to have yeast strains available that do not lose their stress resistance during fermentation . Furthermore, human food habits have changed in the past few years and there has been an increasing usage of frozen dough for bread production. Yeasts under such stress conditions reduce fermentation performance, compromising product quality [36, 99]. Namely, reduced yeast vitality after freezing and thawing the dough causes loss of fermentation capacity and makes it necessary to use higher yeast amounts and longer proofing times (i.e., the resting period after mixing during which fermentation takes place), consequently decreasing product volume . As a result, these effects have a great technological and economic impact in the baking industry. Undoubtedly, the ability of baker’s yeast to cope with stress conditions is an essential physiological requirement in this industry, which evidently would greatly benefit from the availability of yeast strains with improved freeze resistance.
One of the first stresses encountered by baker's yeast cells during the preparation of frozen dough is the cold-shock, i.e. the decrease in the environment temperature after mixing. This change impairs the correct functioning of both the membrane and the translational apparatus as a result of reduced membrane fluidity and stabilization of the secondary structures of DNA and RNA [101, 102]. While positive cold temperatures lead to the synthesis of specific proteins associated with the development of transient phenotypic adaptation [103, 104], freezing is frequently a lethal stress to cells. At sub-zero temperatures, the damaging effects on yeast cells depend on the freezing rate. In the case of rapid freezing, cells are injured by the formation of intracellular ice crystals, which leads to membrane disruption . Structural examination of these cells shows discontinuous nuclear membranes, disappearance of vacuoles, and spreading of DNA all over the cells . On the other hand, in cells exposed to low freezing rates, osmotic shrinkage of the cells and frozen extracellular water is observed. Therein, cells become exposed to hyperosmotic solutions and try to compensate by moving water across the membranes . In this case, cells suffer cellular damage similar to that caused by dehydration. During frozen storage, the growth of ice crystals can further deteriorate the plasma membrane and damage the activity of different cellular systems. Taken together, these findings indicate that freezing is a very complex stress, in which different stress responses appear to play important roles. Therefore, freezing tolerance likely involves different mechanisms working in concert.
The high freezing resistance of T. delbrueckii PYCC 5321 and 5323 strains was the main characteristic that set them apart as potential candidates for the baking industry. In fact, even after freezing these T. delbrueckii cells at –20 ºC for at least 120 days, they retained nearly 100% cell viability, estimated as colony forming units (CFU). In contrast, viability of a Saccharomyces cerevisiae commercial baker's yeast was less than 20% viability by the 15th day . The resistance of T. delbrueckii was due to its ability to maintain the integrity of the plasma membrane, and it diminished in the presence of cycloheximide in the freezing medium. Membrane integrity, evaluated by flow cytometry with propidium iodide staining, correlated directly with the CFU counts for both yeasts, validating the utilization of flow cytometry to measure viability of yeast cells subject to freezing stress. The ability of T. delbrueckii to preserve plasma membrane integrity during freezing does not correlate with the concentration of intracellular trehalose (Tin) at the time of freezing, since the values of Tin were high and in the same order of magnitude in both T. delbrueckii and S. cerevisiae strains. In addition, T. delbrueckii was able to retain much higher cell viability when subject to a period of fermentation before freezing. Under those circumstances, there was also no correlation between membrane integrity and Tin. During the period of prefermentation, the concentration of Tin fell at a high rate and to similar values in both strains, consistent with a similar pattern of activation of trehalase (s) by glucose, determined in cellular extracts of T. delbrueckii and S. cerevisiae. In contrast, the higher capacity to preserve the plasma membrane observed in T. delbrueckii seems to be related to a smaller increase of lipid peroxidation during the freeze storage period. These results suggest that the ability of T. delbrueckii to avoid damage from oxidative stress in the plasma membrane during freezing can contribute to its freeze-resistant phenotype. In agreement with these results, which seem to imply oxidative damage is involved in the loss of plasma membrane integrity during freezing, pre-treatment of S. cerevisiae cells with the radical scavenger N-tert-Butyl-α-phenylnitrone (PBN) led to a reduction in the loss of membrane integrity.
6. Gene disruption in Torulaspora delbrueckii
The use of gene deletion mutants is an important tool to decipher the role and physiological relevance of the proteins encoded by different genes. Results from these studies can increase the knowledge of the physiology, biochemistry and molecular biology of an organism. To this purpose, construction and analyses of Torulaspora delbrueckii mutant strains are of highest importance; however, the genetic tools available for this yeast are very scarce. Disruption of genes followed by phenotypic analyses is a vital tool for understanding yeast gene function. However, the efficiency of gene disruption is highly variable among species, and is often quite low for non-conventional yeasts. In yeast, gene disruption is usually accomplished by transforming cells with a gene-targeting section (cassette) containing a selectable marker, conferring drug resistance or nutrient autotrophy, flanked by upstream and downstream sequences of the gene of interest . These cassettes are frequently generated by PCR, using primers containing both bordering regions of the target gene and part of the selectable marker gene, and subsequently used to transform yeast cells through various transformation protocols, but usually by the lithium acetate TRAFO method . There has been widespread use of gene disruption cassettes generated by PCR [110, 111], since very short sequences of yeast DNA flanking the marker gene are sufficient for efficient integration into the Saccharomyces cerevisiae genome by homologous recombination . Therefore, it is possible to screen a relatively small number of the transformants growing on the selection media (usually by PCR) and confirm correct integration of the cassette. When recombination efficiency is very low but cells are under selection pressure, cassettes are often integrated in the wrong locus, giving rise to a large number of false positives (cells that are able to grow on the selection media, but not actual disruptants), and the number of true positives may be lower than 1 in a 100. It then becomes necessary to perform a secondary screen, which is only possible when deletion of the gene of interest results in a readily identifiable phenotype. Evidence of the low efficiency of T. delbrueckii homologous recombination emerged when disruption of the TdMAL11 gene was attempted ; however, in that case it was possible to screen the transformants through phenotypic analysis, since TdMAL11 null mutants exhibit deficient growth on medium containing maltose as the sole carbon source. However, it was not possible to use the same strategy to disrupt LGT1, the first gene identified as coding for a hexose transporter in T. delbrueckii, since there was evidence that other hexose transporters exist in this yeast . Therefore, we could not screen for potential T. delbrueckii LGT1 disruptants by searching for a clear-cut phenotype, because loss of LGT1 might be compensated by the activities of other genes and was thus not expected to impair glucose growth capacity. Therefore, using the conventional method of transforming a PCR-amplified disruption cassette with a short flanking homology (SFH-PCR) , we were unable to generate ∆lgt1 mutants. Our strategy was thus first to obtain a TdLGT1-targeting cassette harboring longer homology arms, and then to attempt further optimization of the yield of LGT1 disruption, by testing how different parameters inherent to the lithium acetate method described by  contributed to the transformation efficiency . We constructed a cassette with longer flanking regions by inserting a marker-resistance module into the core of the LGT1 gene, and then used this construction as a template for PCR amplification of a TdLGT1 long-flanking homology disruption cassette (figure 2]. Then, to further improve the yield of LGT1 disruption, we reformulated some parameters of the transformation method. Mainly, after heat shock, cells were pelleted and resuspended in rich medium supplemented with low concentrations of geneticin [100 mg mL-1] and incubated overnight (instead of the usual 4-h recovery time). Finally, cells were plated onto selective YPD plates supplemented with higher concentrations of geneticin [300 mg mL-1], to select against false positives, and incubated for up to 4 days.
Integration efficiency using this strategy was extremely high when compared with the conventional method (no disruptants from using the conventional method and 12/16 using this improved method). We thus concluded that two important modifications were the most relevant for our global strategy: the size of the disruption cassette and the new recovery period of cells during the transformation protocol. As a result, this method demonstrated to be a valuable alternative to the conventional PCR-based gene disruption for the yeast T. delbrueckii. This methodology could also be advantageously applied to other non-conventional yeasts, where correct gene disruption with the commonly used short flanking homology cassettes is frequently very low.
7. Molecular characterization of Torulaspora delbrueckii strains
Before we can exploit the potential of Torulaspora delbruecckii in industrial processess, we must be able to identify it and distinguish between strains, using reliable techniques. The accessibility of typing techniques that enable a rapid and accurate differentiation at the strain level is imperative for both wine and baker’s yeast users and producers, to assure that the commercialized yeast corresponds to the strain selected originally. Thus, developing practical typing techniques that enable discrimination between T. delbrueckii strains is an essential tool for its implementation in the baking and wine industries.
In order to determine the suitability of mitochondrial DNA restriction analysis for T. delbrueckii strain differentiation, we selected additional autochthonous yeast strains from the yeast flora present on the home-made corn and rye bread doughs in the northern area of Portugal (unpublished results from our laboratory). We first screened 134 isolates by restriction pattern analysis of both PCR-amplified 5.8S rRNA gene and internally transcribed spacers ITS1 and ITS2, as previously described , selecting only T. delbrueckii species. The total length of the ITS1-5.8S-ITS2 regions of the 5.8S rRNA gene is identical for all T. delbrueckii strains, and for that reason this method cannot discriminate at the strain level [45, 115-117]. Three isolates [45A, 45D and 62C) were selected and placed in the CBMA yeast culture collection, Department of Biology, University of Minho, Braga-Portugal. To discriminate between T. delbrueckii strains, both mitochondrial DNA restriction fragment length polymorphism and pulsed-field gel electrophoresis (PFGE) were applied to the three selected isolates, to T. delbrueckii PYCC 5321 and PYCC 5323, and to type strain ISA1082 (Portuguese Yeast Culture Collection, Institute Gulbenkian de Ciência, Oeiras –Portugal) for a comparative pattern.
Mitochondrial DNA restriction fragment length polymorphism (mtRFLP) analysis has been widely applied to the characterization of reference and commercial Saccharomyces cerevisiae wine yeast strains [118-123], as well as strains belonging to other species [116, 124]. Not all the enzymes used in this method detect the same degree of polymorphisms, which depend greatly on the species. mtRFLP using HinfI is associated with the detection of a high polymorphism and is a widely used genetic marker to distinguish S. cerevisiae wine strains [118, 122, 125, 126]. On the other hand, GC clusters of the mitochondrial genome are the main source of the polymorphisms, and a large portion of these contains restriction sites for Haelll.
For mtRFLP, DNA was isolated from yeast cells grown in YPD and digested with HinfI or HaeIII restriction enzymes. Restriction fragments were separated in horizontal agarose gels (figure 3]. mtRFLP's of the six strains using HinfI or Haelll resulted in two distinct profiles, with slight variability. The major difference was found in the upper bands, where the resolution is better (figure 3 arrows). Apart from these bands, the pattern of the profiles provided by each enzyme is identical, indicating that these strains are genetically very closely related. Restriction with HinfI resulted in one profile including T. delbrueckii ISA1082 (type strain), T. delbrueckii PYCC 5321, 45A and 45D, and in another profile including T. delbrueckii PYCC 5323 and 62C strains (Fig.3A). Restriction analysis with HaeIII resulted in a profile including T. delbrueckii PYCC 5321 and 45A, and a second profile that includes T. delbrueckii ISA1082, T. delbrueckii PYCC 5323, 45D, and 62C strains (figure 3B). This method is therefore not suitable to discriminate between T. delbrueckii strains.
Karyotype analysis is a highly efficient technique to differentiate strains of S. cerevisiae, and was applied by numerous authors to characterize reference and commercial yeasts belonging to different species [118, 120, 122, 123, 127]. The electrophoretic karyotypes of the strains under study were therefore also compared. Intact DNA for pulsed field gel electrophoresis (PFGE) was prepared in plugs as previously described  and PFGE was run in a CHEF-DRII Chiller System (Bio-Rad, Hercules, CA). Under the conditions used, six chromosome bands were detected in all the strains, which is in agreement with previous studies indicating T. delbrueckii has six chromosomes . PFGE gel electrophoresis revealed that the chromosomal DNA banding profiles of the strains differ substantially (figure 4], and six different karyotypes could be defined on the basis of the size of putative chromosomes, thereby allowing the discrimination of 4 strains that were not indistinguishable by mtRFLP.
The different karyotypes of the six strains are consistent with their different phenotypes. Indeed, T. delbrueckii PYCC 5321, PYCC 5323, and T. delbrueckii ISA1082 (type strain) have already been established as different strains  and several physiological and biochemical studies of the other three isolates indicated they also correspond to different strains (our unpublished results). Contrary to reports of molecular typing of other yeasts , where both methods allowed discriminating strains in a similar manner, our results show that karyotyping analysis displayed a much higher discriminative power than mtRFLP for T. delbrueckii strains. A reasonable explanation for this difference may be the need for higher stability of an intact mitochondrial genome in this species than in S. cerevisiae. For instance, we have already shown that the relative contribution of respiration to sugar catabolism is higher in T. delbrueckii than in S. cerevisiae . Although mitochondrial genomes contain a very similar set of genes common to all organisms, mtDNA molecules among species are extremely variable in size and organization . Furthermore, the stability of the mitochondrial genome can be evaluated by the ability to form petite mutants. S. cerevisiae spontaneously produces these mutants, which are deficient in the capacity to respire aerobically. The petite phenotype is correlated with gross alterations and extensive deletions or loss of mtDNA [131, 132]. On the contrary, T. delbrueckii is a petite-negative species, as it doesn’t have the ability to form these respiratory mutants even after prolonged treatment with ethidium bromide [133-135]. The high resolutive capability of the CHEF technique allowed us to differentiate between strains isolated from the same environment and that could not be distinguished by mtRFLP. These results underline this technique as a powerful tool for T. delbrueckii strain differentiation, although there are some factors that limit its applicability, since it is complex and time-consuming and not suitable as a routine technique for strain identification.
In summary, the karyotyping profiles and RFLP’s of mitochondrial DNA of the T. delbrueckii strains PYCC 5321 and PYCC 5323 were clearly different. These data corroborate and complement the results obtained in the past by the classical biochemical methodology , representing an update to the understanding of T. delbrueckii populations present in bread doughs. Furthermore, the availability of functional typing tools that enable differentiation at the strain level is extremely important to the bread and wine industries, to assure traceability of the selected strains.
The biotechnological interest in Torulaspora delbrueckii has increased in recent years due to its particularly high freezing and osmotic tolerance [4, 5, 46]. These features made this yeast species a candidate of potential value for the baking industry. However, the existing knowledge on this yeast is still far from the vast knowledge on the traditional baker’s yeast Saccharomyces cerevisiae. Therefore, studies have been developed to gain insight into the physiology, biochemistry, and molecular genetics of T. delbrueckii.
While two of the most important traits for large-scale baker’s yeast production are its growth rate and biomass yield on sucrose, its leavening ability depends mainly on its capacity to ferment maltose. The pattern of sugar utilization and regulation also determines the yeast capacity to rapidly adapt when changing from sucrose-rich growth medium to the dough. Physiological and biochemical studies of T. delbrueckii in batch cultures with the sugars present in molasses and in bread dough, both alone and in mixtures, showed that T. delbrueckii behaves very similarly to S. cerevisiae with respect to sugar utilization and regulation patterns. However, this yeast modulates respiratory metabolism under aerobic conditions more efficiently, an asset for large-scale production of the yeast. Furthermore, comparative analysis of specific sugar consumption rates and transport capacities suggested that it is the transport step that limits both glucose and maltose metabolism.
So far, only one glucose transporter has been identified in T. delbrueckii, the low-affinity glucose transporter LGT1 . Southern blot analysis of the T. delbrueckii genome revealed the existence of several genes with high similarity to LGT1, suggesting there are several hexose transporters in this yeast, which hampered disruption of the LGT1 gene. The existence of several hexose transporters had first been suggested by the isolation of several plasmids from a genomic library of this strain that could complement the glucose growth defect of the S. cerevisiae hexose transport-null mutant . Despite the phylogenetic closeness of T. delbrueckii and S. cerevisiae, the differences observed between the two species show that the behavior or even the methods that can be applied to the former yeast cannot always be inferred from those of S. cerevisiae. For instance, when we attempted to disrupt the T. delbrueckii LGT1 gene, the current methods used for S. cerevisiae were not suitable, and an optimized disruption method had to be developed.
In modern food technology, traceability is a crucial requirement, and thus establishing a rapid method to discriminate between T. delbrueckii strains is of upmost relevance. This technique would enable correct identification of the inoculated strain from the remaining yeast flora present in the bread dough. In the last years, several methodologies of typing based on DNA patterns have been developed which allowed discriminating closely related yeast strains. In this chapter, two different genetic fingerprinting techniques (karyotype analysis and mtDNA restriction analysis) were presented for detailed genotyping of T. delbrueckii strains. Mitochondrial DNA restriction analysis was not a good technique to differentiate among T. delbrueckii strains isolated from the same ecosystem and genetically very closely related. Chromosome separation by pulsed-field electrophoresis revealed considerable variability in the chromosomal constitution of the strains studied, and turned out to be a useful method to discriminate among T. delbrueckii strains. However, this method of chromosome karyotyping may be too complex, laborious, and time-consuming for the analysis of numerous yeast isolates, in contrast with mtDNA restriction analysis.
Nowadays, yeast strains used in bread industry are involved in large-scale processes and hence are exposed to more extreme stress conditions. On the other hand, development of new products and more versatile processes also require yeast strains with new traits. This chapter aimed to highlight some of these emergent problems/needs in the wine and bread-making industries, including selecting, characterizing, and constructing resistant yeast strains, and strains with important qualities for application in the baking and wine industries, as is the case of some studied strains of T. delbruekii.
Despite the accomplishments reported in this chapter, many important questions remain to be answered regarding sugar transporters and freezing resistance in T. delbrueckii. How many hexose transporters are present in T. delbrueckii? What are their affinities and regulation? Is T. delbrueckii similar to K. lactis, as speculated by Alves-Araújo , based on comparison of sequencing data and regulatory studies of LGT1 expression? Or is this yeast more comparable to S. cerevisiae as is suggested by their similar sugar utilizations patterns ? Evidently, it would be important to continue the characterization of T. delbrueckii strains, as their biotechnological potential has already been established [5, 7, 46]. It is clear that answers to these questions may only arise from future studies. Characterization of T. delbrueckii at the different levels will narrow the gap towards its industrial exploitation and increase knowledge on the so-called non-conventional yeast species.
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