NO174214B - Transformed yeast, yeast types using the transformed yeast as well as the use of the transformed yeast - Google Patents

Abstract

New yeast strains providing for an enhanced rate of the fermentation of sugars, and a process to obtain such yeasts and the use of these yeasts. Yeasts capable of improved fermentation of sugars, a process to obtain these yeasts and the use of these yeasts are provided. The yeasts show higher rates of metabolism resulting in for example higher carbon dioxide and ethanol production in media containing sugars, such as maltose, as main carbon and energy source. The fermentation rate of sugars is improved by the introduction into a yeast of one or more DNA constructs comprising at least one gene encoding a protein promoting the uptake and/or initial metabolic conversion of a transported sugar substrate.

Description

The present invention relates to a transformed yeast and new yeast types that use the transformed yeast, capable of improving the fermentation of sugars, and further the use of these improved yeast types.

It is well known that yeast strains belonging to, for example, the genus Saccharomyces are capable of fermenting sugars to approximately equimolar amounts of CO2 and ethanol under anaerobic conditions. The leavening activity of yeast in dough is a result of this fermentation. The commercial product, baker's yeast, is available in various formulations including pressed yeast or fresh yeast and dry yeast. Dry yeast is available as an active dry yeast and as an instant dry yeast with a moisture content of approx. 6-8$, 3-6$ respectively.

One of the early steps in the metabolism of sugars by the action of yeast is the transport of the sugar molecules across the plasma membrane. Specific carriers for different sugars are expressed in yeast. The uptake of maltose is, for example, dependent on a specific maltose permease. This carrier can exist in two forms that differ in their maximum velocity (Vmax) and affinity constant (Km) (A. Busturia and R. Lagunas, "Biochim. Biophys. Acta", 820, 324 (1985)). The translocation of maltose across the yeast plasma membrane is linked to the electrochemical proton gradient in this membrane. For each maltose molecule taken up, a proton is symported (R. Serrano, "Eur. J. Biochem.", 80, 97 (1977)).

Intracellularly, maltose is hydrolyzed to two molecules of glucose in a reaction that is catalyzed as maltase (α-glucosidase). Glucose is then converted to carbon dioxide and ethanol via the Embden-Meyerhof pathway. Compared to the fermentation of glucose, two additional enzymes are required for the fermentation of maltose, respectively maltose permease and maltase. The synthesis of these enzymes is induced by maltose and is expressed by glucose, fructose or mannose. In non-sugary

("lean") doughs, maltose is the most abundant sugar available to the yeast. If sucrose is added to the dough, this disaccharide is hydrolyzed extracellularly by yeast to glucose and fructose. These hexoses are then taken up by yeast under the influence of distinct permeases.

It is generally found that the addition of sucrose to media containing maltose, for example dough, inhibits the metabolism of maltose by yeast cells. This is due to the fact that the transcription of genes encoding maltose permease and maltase is repressed by glucose (R.B. Needleman, D.B. Kaback, R.A. Dubin, E.L. Perkins, N.G. Rosenberg, K.A. Sutherland, D.B. Forrest and CA. Michels, "Proe. Nati. Acad. Sei .” USA 81, 2811 (1984)).

Genes required for the uptake and hydrolysis of maltose are clustered in a MAL locus (R.B. Needleman et al., supra). Strains of Saccharomyces may contain up to five MAL loci (MAL 1-4 and MAL 6), which are unlinked and located at the telomeres of different chromosomes (J.J. Celenza and M. Carlson, "Genetics", 109. 661-664 (1985) ). A MAL locus comprises genes encoding maltose permease, maltase, and one or more regulatory proteins (MAL regulator) required for maltose induction (R.B. Needleman et al., supra; J.D. Cohen, M.J. Goldenthal, T. Cow, B. Buchferer and J. Marmur, "Mol. Gen. Genet.", 200. 1 (1985 ); R. A. Bubin, E. L. Perkins, R. B. Needleman and CA. Michels, " Mol. Cell. Biol. ii 6, 2757 (1986)) . The genes have been isolated and cloned (A.O. J.D. Cohen et al., supra; R.B. Needleman et al., supra; E.J. Federoff, J.D. Cohen, T.R. Eccleshall, R.B. Needleman, B.A. Buchferer, J. Giacalone, and J. Marmur, "J. Bacteriol. .", 149, 1064 (1982)).

As mentioned above, yeast fermentation in lean dough depends on maltose as the main substrate. Maltose is produced in the dough from starch with the help of amylases which are usually present in the flour. In addition, the flour contains a variable amount (0-0.5$) of free sugars such as glucose, raffinose and so on (H. Suomalainen, J. Dettwiler and E. Sinda, "Process Biochem.", 7, 16 (1972)) . These sugars are quickly consumed by the yeast. Various studies have been published investigating the possible correlation between maltose fermentation and the leavening effect of baker's yeast. In some cases, a positive correlation was found between the rate of maltose fermentation and the activities of maltase and maltose permease. However, a positive correlation between the activities of maltase and maltose permease with the rising ability of lean dough could not be observed (P. Hautera and T. Lovgren, "J. Inst. Brew.", 81, 309 (1975); T. Lovgren and P. Hautera , "Eur. J. Appl. Microbiol.", 4, 37

(1977)).

Transformation of yeast cells with multicopy plasmids containing genes encoding maltase and maltose permease gave a fourfold increase in specific activity of maltase, but maltose permease activity was not enhanced. Introduction of extragenes encoding the regulatory protein resulted in a moderate increase in the specific activity of maltase, but here again no effect on maltose permease activity was observed (J.D. Cohen et al., supra). Obtained transformants were not analyzed for carbon dioxide or ethanol production by these researchers. Thus, the prior art refrained from carrying out such experiments because it had repeatedly been shown that there was no correlation between the activities of maltose permease and maltase and the carbon dioxide production (raising effect) in lean dough (H. Suomalainen, J. Dettwiler and E. Sinda, supra ; H. Suomalainen, "Eur. J. Appl. Microbiol.", 1, 1 (1975); P. Hautera and T. Lovgren, supra; T. Lovgren and P. Hautera, supra).

It has now been found that yeast strains transformed by integrative plasmids, examples of which will be described below, show an enhanced level of maltose permease and maltase activity, compared to the non-transformed strain. These enhanced maltase and maltose permease activities surprisingly coincide with an increase in CC^ production or raising activity, which was also observed in yeast transformed with episomal vectors.

These improved yeast types show higher degrees of metabolism resulting in, for example, higher carbon dioxide and ethanol production in media containing sugars, such as maltose, as the main carbon and energy source. The methods provided involved the use of recombinant DNA techniques in such a way that the rate of maltose fermentation for these yeast types is drastically increased regardless of the presence of other sugars such as glucose.

Furthermore, these improved types of yeast show an excellent leavening activity (gas production) in dough. Analogously, the higher the rate of ethanol production for these yeast types, this will result in a reduced fermentation time for yeast used in the production of potable or industrial alcohol or a larger amount of alcohol produced during a certain period of time.

Furthermore, in the case of accumulation of maltose-inhibiting enzymes which convert sugars or starch, rapid removal of maltose is advantageous for the fermentation process. By the principle of the invention, a yeast is obtained with additional genes encoding maltase and/or maltose permease.

The invention provides a transformed yeast which is improved with respect to the fermentation rate of sugars, and comprises at least one, preferably the homologous, DNA construct comprising at least one gene in the yeast which encodes a protein which promotes the uptake and/or initial metabolic transformation of a transported substrate, the gene is capable of expression in yeast.

According to this, the invention relates to a transformed yeast of the genus Saccharomyces, preferably Saccharomyves cerevisiae, and this yeast is characterized by the fact that it is capable of improved production of carbon dioxide compared to a non-transformed origin of the yeast by fermentation in a medium comprising maltose, where the maltose is fermentable both by the source and the transformed yeast, and that the transformed yeast comprises a DNA construct comprising at least one gene capable of expression in the transformed yeast which codes for a maltose permease, maltase or a maltose regulatory protein.

The degree of fermentation of sugars can improve during several phases of the rise, for example an increased rate caused by the fermentation of maltose or sucrose.

Homologous DNA refers to DNA originating from the same yeast genus. For example, Saccharomyces is transformed with DNA derived from Saccharomyces. In this way, it is possible to improve already existing properties for this yeast genus without introducing new properties, which were not present in this genus beforehand. The improvement of the fermentation rate of sugars can be achieved under aerobic or anaerobic conditions. The genes of interest include permeases, especially maltose permease, saccharidases, especially maltase, kinases and especially hexokinases and glucokinase and the like. The invention can for example be used for a carrier protein which is necessary for the uptake of glucose or fructose and hexokinases and glucokinase which catalyze the initial intracellular metabolic conversion of these hexoses into hexose phosphates.

There are effective methods for introducing at least one homologous DNA construct into the yeast.

These can be applied to a construct which comprises at least one gene which codes for a protein which promotes the uptake of maltose and the initial metabolic conversion of maltose into glucose. In this way, yeast can be obtained which has several advantages compared to the original (host) strain. The advantages of such improved yeast types can be found especially in the improved ethanol and COg production. When applied to baker's yeast, this will be a huge advantage to a baker because he needs less time or less yeast to develop lean dough due to the improved leavening action of the new strains.

It should be clear that the transformed yeast according to the invention can be used as a starting strain in the strain-improving procedures different from DNA-mediated transformations, for example protoplast fusion, mass adaptation and mutation.

According to a preferred embodiment of the invention, the new strains will consume significant amounts of maltose in the presence of glucose. The baker can therefore also save sugar costs because less sugar is needed to obtain sweet doughs.

It is well known that osmotolerant yeast types show a poor performance (raising effect) in lean dough. By osmotolerant yeast types is meant yeast that has a good performance in sweet doughs. Doughs for sweet baked goods, for example, will contain 10-30$ of sugar, calculated on the weight of flour. When an osmotolerant strain is therefore chosen as host for transformation, an osmotolerant yeast is obtained which can not only be used in sweet doughs, but also in lean doughs because the capacity for maltose fermentation according to the invention is improved. As a consequence, the baker needs a type of yeast both for sweet and for lean dough.

This need for yeast strains that have good performance in lean as well as sweet dough is described, for example, in EP-A-128524 and DE-A-2757778. In order to obtain yeast strains that perform well in sugar-rich and lean doughs, EP-A-128525 describes a proto-plast fusion method: DE-A-2757778 describes, in order to obtain such yeast strains, a selection method of strains from a population of diploid strains prepared by hybridization or mutation. Both procedures require a lot of experimentation and the results are unpredictable and non-reproducible. By using the method described here, controlled and reproducible results can be achieved with the transformed yeast strains. "Testing of the strains produced may be minimal because the properties of the strains themselves are essentially unchanged apart from the described improved properties.

The invention also relates to pressed, instant-dry or active dry yeast which is characterized by the fact that it has been obtained from a transformed yeast as described above.

The present invention thus provides a pressed yeast which shows a gas production of at least 340 ml/285 mg dry weight of yeast during 165 minutes in sample B and a gas production of at least 170 ml/285 mg dry weight of yeast in sample B'. Samples B and B' are described below. Preferably, the pressed yeast shows a gas production of 380-450 ml/285 mg dry weight of yeast during 165 minutes in sample B and 180-240 ml/285 mg dry weight of yeast in sample B' and most preferably 400 ml/285 mg dry weight of yeast in sample B and respectively 190 ml/285 mg dry weight of yeast in sample B'. Advantageously, instant dry or active dry yeast is produced from this pressed yeast. During drying of the pressed yeast, approx. 15-25$ raising activity, calculated on dry matter, lost.

The invention thus also provides a dry yeast with 3-8 weight-$ moisture and which shows a gas production of 310-360 ml/285 mg dry weight yeast for 165 minutes in sample C and 145-195 ml/285 mg dry weight yeast in sample C and aller preferably 330 ml/285 mg dry weight yeast in sample C and respectively 155 ml/285 mg dry weight yeast in sample C. The gas values obtained with yeast prepared as described here have never been achieved before even when commercially available strains were tested in samples C and C '.

It is also possible to obtain yeast types that show a good performance in raising activity in the range of 0-6 or 0-10$ sugar dough. In this way, it is possible to produce pressed yeast types that show a gas production of 400-500 ml/285 mg dry weight of yeast during 165 minutes in sample B, preferably these pressed yeast types show a gas production of at least 440 ml/285 mg dry weight of yeast. The dry yeast can then be obtained and which shows a gas production of 320-400 ml/285 mg dry weight of yeast during 165 minutes in sample C, preferably the dry yeast shows a gas production of at least 350 ml/285 mg dry weight of yeast.

Corresponding advantages for these new strains are found in fermentation for the production of potable and industrial alcohol.

Because the overall metabolic rate of these new strains is increased when maltose serves as substrate, the overall production rate of metabolites such as glycerol and aroma compounds may also be increased.

Within the teachings of the invention, vectors are described which carry a DNA construct which encodes one or more proteins involved in maltose fermentation. The invention also provides a microbial host, preferably a yeast, which is for example a species of Saccharomyces transformed with vectors as described. These vectors may be self-replicating and preferably contain a gene or a combination of genes selected from those encoding maltose permease, maltase and maltose regulatory protein. Surprisingly, it has been found that yeast transformed with such vectors show an improved rate of maltose fermentation, resulting in an increased rate of (^-production in dough. However, these additional genes are located on episomes and it is known from the literature that such extra chromosomal molecules are easily lost during non-selective propagation (that is, growth in the absence of G418 in this particular case) (CD. Hollenberg

(1982) "Gene Cloning", 12, "Organisms other than E. coli", ed. P. H. HofSchneider, W. Goebel, Springer Verlag, 119; SO. Parent, CM. Fenimone and K.A. Bostian (1985), "Yeast", 1, 83). From a practical point of view it is preferred to grow the yeast non-selectively and therefore a set of integrating plasmids containing preferably altered maltase and/or maltose permease genes is constructed. By "altered" is meant replacement of the natural promoter with another promoter, preferably homologous. In the event that processes are developed that allow stable proliferation of plasmids in the absence of a selective pressure, integration of newly introduced DNA is no longer necessary to obtain stable transformants.

It is well known from the literature that a cell contains between 20 and 100 molecules of these additional plasmids (CD. Hollenberg (1982), supra; A. Takagi, E.N. Chun, C. Boorchird, S. Harashima and Y. Oshima, "Appl. Microbiol. Biotechnol." 23. 123; J. Mellor, M.J. Dobson, N.A. Roberts, N.J. Kingsman and S.M. Kingsman (1985), "Gene", 33» 215).

The expression level of episomal genes can be further increased by replacing the original promoters with stronger promoters. It seems likely that in the future it will be possible to allow stable replication of plasmids in the absence of a selective pressure.

According to the invention, the maltase and/or maltose permease genes are advantageously integrated into the yeast chromosome via transformation with linear plasmids (see T.L. Orr-Wever, J.W. Szostak, R. Rothstein (1981), "Proe. Nati. Acad. Sei." USA 78, 6354 ). The yeast types obtained are stable transformants, that is to say that altered maltase and/or maltose permease genes can be maintained in the genome even in the absence of selective pressure. It is known that during integration only one or a few copies of genes located on a plasmid are integrated into the chromosome. In order therefore to achieve a similar improvement in the CC^ production which is achieved when episomal vectors are used, the gene expression level can advantageously be changed by using strong constitutive promoters, such as are not sensitive to glucose repression. Such promoters are preferred to compensate for the difference in gene copy number of yeast transformed with episomal and with integrative vectors, and to prevent the effect of glucose repression. For example, it has been observed that during the first 30 to 40 minutes of fermentation in media containing maltose as the main carbon and energy source, and relatively low concentrations of glucose, the mRNA levels of maltose permease and maltase decrease to almost undetectable levels. Once the glucose is consumed, the induction of gene expression by maltose results in a rapid increase in both mRNA levels.

As indicated, the genes may be used with their natural or wild-type promoter or the promoter may be replaced with another promoter, preferably a homologous promoter. Where the wild-type promoter is regulatable or inducible, it may be particularly desirable to provide a constitutive transcription, or a stronger or weaker promoter. Where the reverse wild-type promoter is constitutive, it may be of interest to provide a regulatable or inducible promoter or a stronger or weaker promoter.

Strong promoters are preferably used, especially where there may be a relatively low copy number of the construct in the host. Strong promoters will usually be those involved in the production of proteins that are produced at high levels during the life cycle of the yeast or where they are regulatable, during a certain period of interest in the life cycle of the yeast, related to the invention.

Promoters associated with the glycolytic cycle of yeast are of particular interest, which include alcohol dehydrogenase I and II, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, enolase, phosphoglyceromutase, pyruvate kinase lactate dehydrogenase. Other promoters involved with proteins produced in high amounts include promoters associated with *ribosomal expression such as promoters for transcription of initiation factors, elongation factors and the like. Special elongation factors include EF-1 and EF-2, and so on. ;Of particular interest is the use of promoters in combination with structural genes involved with maltose metabolism, particularly maltase, maltose permease and MAL regulator. These promoters can be constitutive or regulatable, as long as the promoter is induced during fermentation of the sugar. For example, many of the glycolytic promoters are activated in the presence of a sugar or a sugar metabolite such as ethanol. Thus, promoters such as alcohol dehydrogenase will be active during rising of the dough. Similarly, the promoters associated with cell proliferation will also be active during dough rising. Furthermore, and by providing promoters that are not regulated by the MAL regulator, the dough can be used in the presence of glucose without repression. In addition, the genes do not require maltose for induction. Where wild-type promoters are used in connection with the structural genes of interest, it may be desirable to ensure increased production of a regulatory protein. In this way, the regulatory protein can be maintained at a high level when the inducer is present. In the presence of maltose, for example, the MAL-regulatory protein will be expressed at a high level to give expression of the other proteins associated with maltose metabolism and regulated by the MAL-regulatory protein. ;The changes in gene expression as described in detail in the experimental procedures below include the substitution of the original promoters plus (part of) the untranslated leader sequences for those of alcohol dehydrogenase I (ADHI) and the translation elongation factor EFlaA which is preferably derived from the host yeast, for example Saccharomyces. As a consequence, the expression will become insensitive to glucose repression and independent of maltose for induction. It has been found that yeast strains transformed by these integrative plasmids show an increased level of maltose permease and maltase activity, compared to the non-transformed strain. These increased maltase and maltose permease activities surprisingly coincide with an increase in CC^ production or raising activity as was also observed in yeast transformed with episomal vectors. ;The achieved improvement in the raising activity is maintained during storage even at elevated temperatures, for example at 20-25°C. The relative loss of heaving activity during storage is essentially identical for the parent strains and the strains according to the invention. The raising activity of hay sugar doughs is not affected by the manufacturing process because the raising activity of the new strains transformed with integrative plasmids is as good as that obtained with the host strain. ;The present yeast host will have at least one copy of the construct and may have two or more, usually no more than approx. 200, depending on whether the gene is integrated into the genome, amplified or present on an extra chromosomal element with multiple copy numbers. Integration or non-integration can be selected depending on the stability required for maintaining the produced extrachromosomal element, the desired copy number, the level of transcription available depending on the copy number and the like. The construct may include one or more structural genes with the same or different promoters. The construct can be produced in conventional ways, by isolating the desired genes from a suitable host, by synthesizing all or part of the genes or combinations thereof. Similarly, the regulatory signals, the transcription and translation initiation and termination regions, can be isolated from a natural source, synthesized or combinations of all these. The various fragments can be subjected to endonuclease digestion (restriction), ligation, sequencing, in vitro mutagenesis, primer repair or the like. The various manipulations are well known in the literature and will be used to achieve specific purposes. The various fragments can be combined, cloned, isolated and sequenced according to conventional methods. After each manipulation, the DNA fragment or combination of fragments can be inserted into the cloning vector, the vector transformed into a cloning host, for example E. coli, the cloning host grown, lysed, the plasmid isolated and the fragment analyzed by restriction analysis, sequencing or combinations thereof or the like. ;Different vectors can be used during development of the construct and transformation of the host cell. These vectors may include cloning vectors, expression vectors and vectors that allow integration into the host or the use of pure DNA for transformation and integration. ;The cloning vector will be characterized, at least substantially, by having an origin of replication functional in the cloning host, a marker for selection of a host containing the cloning vector, may have one or more polylinkers or additional sequences for insertion, selection, manipulation, easy sequencing, excision etc. In addition, shuttle vectors can be used where the vector can have two or more origins of replication, allowing the vector to be replicated in more than one host, for example a prokaryotic and a eukaryotic host. The expression of vectors will usually result in the insertion of a construct that includes the transcriptions and translations, the initiation region and the termination region or the construct may lack one or both of the regulatory regions that will be provided for by the expression vector by insertion of the sequence that codes for the protein product. Thus, the construct can be inserted into a gene with the functional transcriptions and translation regions where the insertion is proximal to the 5'-terminus or the existing gene and the construct comes under the regulatory control of the existing regulatory regions. Generally, it will be desirable for the initiation codon to be 5' to the existing initiation codon if a condensed product is not acceptable, or the initiation codon is out of phase with the existing initiation codon. In other cases, there are expression vectors that have one or more restriction sites between the initiation and termination regulatory regions so that the structural gene can be inserted into the restriction site or sites and be under the regulatory control of these regions. Of particular interest for the present invention as a vector for the expression, either for extrachromosomal stable maintenance or integration, are constructs and vectors which, in their stable form in the host, are free of heterologous (non-Saccharomyces) DNA. Yeasts according to the invention which exhibit all the above-described advantages while additionally having prokaryotic DNA sequences removed can be obtained by gene replacement techniques (R.J. Rothstein (1983), "Methods in Enzymology", 101, 202). Accordingly, these techniques are now advantageously applied to Saccharomyces cells. An example is the transformation of Saccharomyces cells with a vector containing genes encoding altered maltase and/or maltose permease located in a Saccharomyces sporulation-specific gene (E. Gottlin-Ninga, D.B. Kaback (1986), "Mol. Cell. Biol. " 6, 2185). After introduction of this DNA into a Saccharomyces host cell, homologous recombination of the newly introduced DNA with the chromosomal sporulation-specific gene occurs. As a consequence, altered maltase and/or maltose permease genes are embedded between the sporulation-specific sequences integrated into the chromosome. Resulting transformants are completely free of prokaryotic DNA. ;It will be clear that even better results can be obtained if the optimum ratio between maltase and maltose permease activity is determined. This can happen by varying the promoters for both genes or by integrating different numbers of (altered) maltase and maltose permease genes into the yeast genome, for example by using different integration loci, according to the methods described below. The optimal ratio between maltase and maltose permease activity can also be achieved by using maltase or maltose permease genes that encode other isoenzymes of maltase and maltose permease. Furthermore, any enzyme can be used which has at least maltase or maltose permease activity. In addition, genes encoding the MAL regulatory protein can be integrated into the genome in an analogous manner (see also example 1), either under their own control or under the control of a natural promoter or under the control of another promoter, preferably Saccharomyces. This can also be useful for achieving an optimal ratio between maltase and maltose permease activity. ;Below follows a list of deposited strains. ;These strains have been deposited at the "Centraal Bureau voor Schimmel-cultures" in Baarn, Holland: Saccharomyces cerevisiae 237 Ng (strain A) has been deposited at CBS under accession number 158.86 on March 25, 1986; Saccharomyces cerevisiae DS 15543 (strain C) has been deposited at CBS under accession number 406.87 on 3 September 1987; Escherichia coli containing plasmid p21-40 has been deposited at CBS under accession number 400.87 on 28 August 1987; Escherichia coli containing plasmid pYEF46 has been deposited at CBS under accession number 401.87 on 28 August 1987; Escherichia coli containing plasmid pY6 has been deposited at CBS under accession number 402.87 on 28 August 1987; Escherichia coli containing plasmid peG418 has been deposited at CBS under accession number 160.86 on 25 March 1986; Escherichia coli containing plasmid pTZ19R has been deposited at CBS under accession number 405.87 on September 3, 1987; Escherichia coli containing plasmid pTZ19R/ÅDHI has been deposited at CBS under accession number 404.87 on September 3, 1987; Escherichia coli containing plasmid pl53-215 ÅK has been deposited at CBS under accession number 403.87 on September 3, 1987. Escherichia coli containing plasmid pLF24 has been deposited at CBS under accession number 156.88 on March 8, 1988. Escherichia coli containing plasmid pUT332 has been deposited at CBS under accession number 158.88 on March 8, 1988. Escherichia coli containing plasmid pTZ18R has been deposited at CBS under accession number 480.88 on July 27, 1988. Brief description of the drawings. Figure 1 describes the construction of plasmid pGb-eMAL69. ;Arrows indicate the direction of transcription for the indicated genes. The plasmids are drawn schematically and not to scale. Abbreviations. G418 = Tn5 gene (under control of ADHI promoter) confers resistance to G418; ;P = Pvul; X = Xbal; S = Sali; H = HindIII; CIP = calf intestinal phosphatase. Figure 2 describes the construction of pGb-eMAL61. The plasmids are drawn schematically and not to scale. Abbreviations: A maltase = partial deletion maltase gene; B = Bgllll. See also the description of Figure 1. Figure 3 shows the construction of plasmid pGb-eMAL63. The plasmids are drawn schematically and not to scale. Abbreviations: (K) = filled Kpnl seat; (S) = filled Sall seat; Klenow = large subunit DNA polymerase; H = Hindlll. See also the description of Figure 1. Figure 4 shows the construction of pGb-M6g(A-9). Abbreviations: B = Hindlll; St = Chair; Klenow = large fragment DNA polymerase I; EV = EcoRV; Xh = XhOI; M = Mlul; fl ori = origin for replication subject fl; amp = ampicillin resistance gene; s.p. = sequence primer. ;The arrows indicate the 5' 3' direction. Excluded regions are indicated by dashed lines and A. Figure 5 shows the sequence of plasmid pGb-M6g(A-9). ;a) maltase and maltose permease genes. The arrows indicate the direction of transcription. St (Stul) served as the starting point for the construction of the deletion mutant. Relevant parts of the sequence of intergenic area are shown below this map. ;b) The sequence of pGb-m6g (a-9). Omitted areas range from -9 to -417. Polylinker; refers to the oligonucleotides ligated to the Bal31-treated DNA (see also Figure 4). Figure 6 describes the construction of plasmid pGb-iA32/G418. ;Arrows indicate the direction of transcription. The plasmids are drawn schematically but not to scale. Abbreviations: H = Hindlll; EV = EcoRV; fl ori = origin of replication phage fl; amp = ampicillin resistance gene; G418 = Tn5 gene (ADHI promoter) conferring resistance to G418; S = Narrow; Hc = Hindi; pADHI = promoter alcohol dehydrogenase I gene + part 5' leader (shaded area). Figure 7 shows the construction of plasmid pGb-iRRO1. ;a) plasmid pT4 is not drawn to scale. Abbreviations: E = EcoRI; B/Bg = BamHI/Bglll ligation; H = Hindlll b) mutagenesis on pT4 to work in EFlaA promoter + 5' leader to the five N-terminal amino acid codons of the maltase gene in such a way that a Bglll site is also formed. The relevant sequences are shown. - The mutagenesis primer is partially complementary (indicated by dots) to the EFlcxA sequence. In the mismatch region are the matase codons and the —boxed— Bglll recognition site (note that the shown orientation of the mutagenesis primer is 3' -> 5', i.e. the Bglll site must be read from right to left (5 -► 3')). - In the maltase sequence, N-terminal 5-amino acid codons are indicated. The Bcll recognition site is boxed. - In the sequence pT4-M, the sequence covering the mutation is shown. The bglll seat is boxed. Apostrophes indicate the deviation from the maltase nucleotide sequence. The deviation in the 4th codon is a silent mutation. c) The plasmids are not drawn to scale. Abbreviations: H = Hindlll; Bc = Bell; E = EcoRI; Bg , = Bglll; EV = EcoRV; Bg/Bc = Bglll/Bcll ligation; pEFlaA (shaded box), 5' flank (promoter + 5' leader sequence) of EFlaA; fl ori = origin of replication for phage fl; amp the ampicillin resistance gene. d) The plasmids are not drawn to scale. ;Abbreviations: See under section c). Figure 8 describes the construction of plasmid pGb-SNENS. ;The plasmids are drawn schematically and not to scale. Abbreviations: E = EcoRI; H = HindIII; Sf = Sfile; N = Note; fl ori = origin of replication for phage fl; amp = ampicillin resistance gene. Arrows indicate the 5' - 3' direction. Oligo 3 and 4 are synthetic oligodeoxynucleotides with the base sequence as indicated. Figure 9 describes the construction of plasmid pGb-RB2. ;The plasmids are drawn schematically and not to scale. Abbreviations: E = EcoRI; Bg = Bglll; H = HindIII; ;Hc = HincII; B = BamHI; Sm = Narrow; P = Pstl; fl ori = origin of replication for phage fl; amp = ampicillin resistance gene. Arrows indicate the 5' -+ 3' direction. Oligo 5 and 6 are synthetic oligodeoxynucleotides with the base sequence as indicated. Underlined are TAA translational stop codons in all reading frames. Figure 10 shows the construction of the plasmid pGb RBN 3. Arrows indicate the direction of transcription. The plasmid is drawn schematically and not to scale. Abbreviations: Sf = Sfil; Sm = Narrow; H = HindIII; fl ori = origin of replication for phage fl; amp = ampicillin resistance gene. G418 = Tn5 gene (under control of ADHI promoter) conferring resistance to G418. EV = EcoRV; ;Hc = HincII. Figure 11 describes the construction of plasmid pGb-RBRRO1. Plasmid pGb-iRRO1 is fully described in Figure 7 and contains the maltase gene under the control of the EFlaA promoter (pEFlaA) and the maltose permease gene under the control of the alcohol dehydrogenase-I promoter (pADHI) (both promoters are indicated by shaded boxes). pGb-RB2 is described in Figure 9. In pGb-RBRROl, the SIT4-containing fragment is divided into two parts ("SIT4 flanks"). The plasmids are drawn schematically and not to scale. The abbreviations are as in Figure 10. Arrows indicate the direction of transcription. ;Figure 12 describes one-step gene disruption. In step 1, pGb-RBN3 is degraded with Sfil. This releases a DNA fragment that has homology on both sides to the SIT4 gene region. This directs integration into the SIT4 gene (see also R.J. Rothstein (1983) in "Methods in Enzymology", 101, 202). The SIT4 gene region is indicated by a shaded box. Both chromosomal alleles are shown schematically. In step 2, the resulting strain ApGb-RBN3 transformed with both pUT332 (non-degraded) and pGb-RBRRO1 is degraded with SfiI. The principle of cotransformation is well documented (cf. A.H. Brand, I. Breeden, J. Abraham, R. Steiglanz and K. Kasmyth (1985), "Cell", 41, 41-48 and P. Siliciano and K. Tatchell (1984) "Cell" 37, 969-978). In the first selection, resistance to phleomycin (plasmid pTJT 332) was used, but of course 2y-derived episomal plasmids can also be used which provide resistance to other antibiotics such as hygromycin B. pUT332 and phleomycin are commercially available from Cayla, Avenue Larrien, Center Commercial de Gros, 31094 Toulouse Cédex, France. On pUT332, the phleomycin resistance gene is derived from transposon Tn5 and is placed under the control of a yeast promoter. In step 3, the episomal plasmid pUT332 is removed from the transformants by cultivation in non-selective medium (maturation). Abbreviations: G418<r> = G418 resistance gene under control of ADHI promoter; Sf = the end of a DNA fragment formed by Sfi degradation; MAL = an altered maltase and maltose permease gene. See also Figures 9 and 11 for details on the plasmids; +pUT322, episomal plasmid pUT332. Figure 13 describes the correlation between the increase in specific activity for maltose permease and maltase with the disappearance of glucose from medium A. The diagrams are characteristic of commercial baker's yeast strains such as strain A. Figure 14 shows the specific activities for maltose permease in strain A and its rDNA derivatives during simulation of a dough rise in medium A. Figure 15 describes the specific activities of maltase during a simulation of dough rise in strain A and its rDNA derivatives in medium A containing maltose as the main carbon and energy source. Figure 16 describes the fermentation of maltose during simulation of dough rise with strain A and its rDNA derivatives in medium A containing maltose as the main carbon and energy source. Figure 17 describes the fermentation of maltose during simulation of dough rise with strain A and its rDNA derivatives in medium B containing glucose as the main carbon and energy source. ;The following experimental data are given to illustrate the invention. It should be clear that the person skilled in the art can use other yeast strains and vectors without going outside the scope of the invention. ;Cloning techniques ;For general cloning techniques, reference should be made to the manual by Maniatis et al. (T. Maniatis, E.F. Fritsch, J. Sambrook (1982), "Molecular Cloning, A Laboratory Manual"). Restriction enzymes are used according to the manufacturer's recommendation and are obtained either from New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Boehringer Mannheim (Boehringer). In general, 1 to 5 enzyme units are required to cleave 1 Mg of DNA. Transformation of E. coli was carried out using the CaClg technique (T. Maniatis et al., supra). ;Construction of recombinant plasmids ;1. pGb-eMAL6g; This plasmid is capable of self-replication in yeast and contains the genes encoding maltose permease and maltase. The construction is outlined in Figure 1. ;peG418 is derived from pEMBLYe23 (Baldari and G. Cesarini ;(1985), "Gene" 35, 27) and contains between the Sali and HindIII sites a fragment with the Tn5 gene (Reiss et al ., "EMBO J." ;(1984) 3, 3317), conferring G418 resistance under the control of the alcohol dehydrogenase I (ADEI) promoter, from yeast, similar to that described by Bennetzen and Hall (J.C. Bennetzen and B. D. Hall (1982), "J. Biol. Chem." 257, 3018). peG418 was cleaved with HindIII, dephosphorylated with CIP and ligated with a digest of pY6 x HindIII x Pvul. pY6 has been described (R.B. Needleman, D.B. Kaback, R.A. Dubin, S.L. Perkins, N.G. Rosenberg, K.A. Sutherland, D.B. Forrest, C. Michels (1984), "Proe. Nati. Acad. Sei." USA, 81, 2811) and contains a 7.0 kb HindiII fragment spanning the MAL6g locus. This gave pGb-eMAL6g. ;2. pGb-eMAL61 ;This 2jj-derived episomal plasmid contains the gene encoding maltose permease. The construction is outlined in Figure 2. ;pGb-eMA16g contains two Bglll sites, both located in the maltase gene. This 1.4 kb BglII is omitted from pGb-eMAL6g by digestion with BglII, followed by dilution religation to promote intramolecular ligation. Such an omission has been shown to destroy maltase function (J.D. Cohen, M.J. Goldenthal, T. Chow, B. Buchferer and J. Marmur (1985), "Mol. Gen. Genet.", 20» 1). ; 3. pGb-eMAL63 ;This 2>j-derived episomal plasmid contains DNA covering the MALp function (regulatory protein gene or MAL regulator). The construction is indicated in Figure 3. From p21-40 (R.B. Needleman and C. Michels (1983), supra) the KpnI-SalI fragment containing the regulatory protein gene was isolated. This fragment was blunt-ended using T4 DNA polymerase and Klenow DNA polymerase and then cloned into the filled HindIII site in peG418. ; 4. pGb-M6g(A-9) ; This plasmid is a promoter deletion mutant produced in the intergenic region of the divergently transcribed genes maltose permease and maltase (see Figure 4). This region contains the promoters for both genes (S.H. Hong and J. Marmur; (1986) "Gene", 41, 75). This deletion mutant is designed to replace the original promoters. The construction includes the following steps: a) At approx. The 7.0 kb HindIII fragment containing the genes for maltase and maltose permease (see also Figure 1) was cloned into the HindIII site of pTZ19R. This plasmid is commercially available from Pharmacia. This results in pGb-M6g. ;b) pGb-M6g was linearized with Stul that cuts in the intergenic region. Stul-generated ends served as the starting point for the exonuclease Bal31 to remove (parts of) the promoter-containing intergenic region. Stul is closer to the maltose permease gene (S.E. Hong and J. Marmur; (1986), supra). Bal31 incubation was carried out as described by Maniatis et al. (T. Maniatis, E.F. Fritsch and J. Sambrook (1982), supra). At appropriate times, samples were removed from the reaction and incubated with Klenow DNA polymerase to create blunt ends. Then, synthetic linkers were ligated to the ends containing multiple restriction sites. ;The following complementary oligodeoxynucleotides were used: ;1. 5' GATATC CTCGAG AGGCCT A 3' ;2. 3' CTATAG GAGCTC TCCGGA TGCGC 5' ;In double-stranded form, restriction sites were formed for EcoRV (GATATC), Xhol (CTCGAG), Stul (AGGCCT), ligation at the sticky end yielded an Mlul site (ACGCGT). After kinase reaction, the linkers were ligated onto the Bal31-treated DNA according to conditions as described (T. Maniatis et al., (1982), supra). The reaction mixture was then incubated with ;MluI and chromatographed through a 5 ml Sepharose C1-2B column to separate unligated oligodeoxynucleotides from the DNA fragment. Fractions containing this linear DNA were pooled, ligated to plasmid DNA and introduced into bacteria. c) The resulting set of deletion mutants was subjected to sequence analysis. For this purpose, the double-stranded plasmids were converted to single-stranded DNA by superinfection with a helper phage (protocol according to the supplier's recommendations). The single-stranded templates were extracted by normal M13 procedures for use in dideoxy sequencing (F. Sanger, S. Nicklen and A.R. Coulson (1977) "Proe. Nati. Acad. Sei." 74, 5463). As a primer, a synthetic oligodeoxynucleotide (5'—GAATTCGGTAGCGTTCACGC-3') was used, complementary to a stretch of DNA near the ATG start codon of the maltose permease gene. For the orientations so that the sequence is read against the promoter, see also Figure 4. The deletion mutant in which most of the maltose permease promoter has been removed was chosen for further experiments. (Part of) the maltase promoter is still present (note that the Stul site as the starting point for exonuclease processing is located asymmetrically in the intergenic region). Figure 5 summarizes the sequence at the point of deletion for the mutant pGb-M6g(A-9). This is compared with the recently determined sequence for this total area (S.H. Hong and J. Marmur; (1986), supra). In the wild-type sequence, a difference is noted from the published sequence: C at -878 (numbering according to S.H. Hong and J. Marmur (1986), supra) is not present in the present sequence. Accordingly, DNA cannot be degraded by HpaI or HincII at this position. ;Plasmid pGb-M6g(A-9) is the starting plasmid for the incorporation of other promoters for both the maltose permease gene and the maltase gene (see below). ;5. pGb- iA32/ G418 ; This plasmid is an integrating yeast plasmid. It contains the maltose spermase gene, linked to the alcohol dehydrogenase-I promoter and part of its 5' leader sequence. The construction was as follows (figure 6). ;a) Plasmid pTZ19R/ADHI contains a 1.4 kb BamHI fragment with the ADHI promoter, starting at position -15 relative to the AUG codon (J.L. Bennetzen and B.D. Hall (1982), "J. Biol. Chem. ", 257. 3018). From this plasmid, a 700 bp EcoRV-HincII fragment was isolated and ligated into EcoRV-digested pGb-M6g(A-9). Resulting plasmids were analyzed by restriction enzyme digestion and the correct orientation was confirmed via dideoxy sequence analysis on single-stranded templates (see Figure 6). The same oligoprimer was used as described in section 4c. ;As a result of the cloning procedure of the ADHI promoter fragment, part of the polylinker of pTZ19R (BamHI-HincII) is present between the maltose permease gene and the ADHI promoter. The structure and sequence of pGb-A32 is shown in Figure 6a. b) Plasmid pGb-A32 was equipped with the dominant selection marker G418<res>. A 1.9 kb EcoRv/HincII fragment containing the Tn5 gene confers resistance to G418 under the control of promoter alcohol dehydrogenase I. This fragment was isolated by an EcoRV/HincII double digestion of plasmid 153-215 AK. This EcoRV/HincII fragment was cloned into the SmaI site of pGb-A32. This gave pGb-iA32/G418. 6. pGb- iRRol ; This plasmid is an integrating plasmid. It contains the maltose spermase gene under the control of promoter alcohol dehydrogenase I and the maltase gene under the control of promoter translation elongation factor EFlaA. The cloning route is indicated in Figure 7. The approach was as follows: The maltase gene contains a Bcll site around the fifth amino acid codon. Therefore, the EFlaA coding region was mutagenized in such a way that the first five amino acids became identical to those of the maltase protein. A Bglll seat was co-introduced in the position of the Bclll seat. Conversion of a Bcll site to a Bglll site is a silent mutation in the fourth codon. Via a Bglll/Bcll ligation, the EFlaA promoter and the leader sequence could be united with the rest of the maltase gene. The procedure included the following steps: a) The starting plasmid was pYEF46 (S. Nagata, K. Nagashima, Y. Tsunetsugu-Yokota, K. Fuyimura, M. Miyaraki and Y. Kaziro; (1984), "EMBO J.", 3, 1825 ) which contains the entire gene that codes for EflaA. A 2.5 kb BglII fragment covering this gene was isolated and cloned into the BamHI site of pTZ19R. The clone pT4 was captured (see Figure 7a) and used for the oligodeoxynucleotide-directed mutagenesis. ;b) After superinfection with helper phage, single-stranded (ss) DNA of pT4 was isolated. 400 ng of ss DNA, 400 ng of heat-denatured pTZ19RxBamHI and 100 ng of mutagenesis oligodeoxynucleotide (see Figure 7b) were incubated in a volume of 10 µl of 7 mM Tris-HCl, pH 7.5; 50 mM NaCl and 7 mM MgCl2 for 10 min at 56°C. After 10 minutes at room temperature, the second strand synthesis and ligation was initiated by the addition of 1 µl of Klenow DNA polymerase (2U). 1 µl of T4 DNA ligase (4U), 1 µl TMD (200 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 100 mM DTT), 4 µl 2.5 mM dNTP mix, 1 µl 10 mM ATP and 2 pl H2O. The final volume was 20 µl, the incubation was carried out for 16 hours at 17°C after which the mixture was transformed into E. coli JM101. Mutants were scored by colony hybridization with a kinased oligodeoxynucleotide, specific for the mutant (see Figure 7b). This judgment oligomer 5'—GACTATTTCAGATCTTC-3' was complementary to the introduced maltase codons and flanking nucleotides. The hybridization was carried out for 16 hours in 6 x NET (1 x NET = 0.15 M NaCl, 0.016 M Tris-HCl, pH 7.5, 0.001 M EDTA) at 25°C. Post-hybridization washes were performed with the same mixture at the same temperature (3 x 10 minutes), followed by a wash in 3 x NET at 25°C. Several positive colonies were analyzed further. BgllI digestion confirmed the presence of a Bglll site. In addition, single-stranded DNA was isolated from a Bglll site-containing mutant (pT4-M) and subjected to dideoxy sequence analysis using a synthetic 17-mer primer, complementary to nucleotides 87 to 103 of the EFlaA gene (S. Nagata, K. Nagashima, Y. Tsunetsugu-Yokota, K. Fuyimura, M. Miyaraki and Y. Kaziro (1984) "EMBO J.", 3, 1825) (5'—CAATACCACCACACTTG-3').The obtained sequence confirmed the successful introduction of the desired mutations. ;c) The next step was to isolate the EFlaA promoter/NB^-terminal maltase gene segment from pT4-M and to join this via ;Bglll/BclI sticky end to the rest of the maltase gene. This step restores the maltase coding region downstream of the EFlaA promoter and the leader sequence (see Figure 7c).Plasmid pGb-M6g(A-9) was transformed into GM113, an E. coli pond strain, to utilize the Bcll site (which is methylation sensitive) (GM113: thr-, leuB6, proA2, tri-4, metBl, lacYl, galK2, ara-14, tsx33, thi- 1, thyA12, deoB16, supE44, rpsL260, dam-3). A 3.8 kb BclI/HindIII fragment was isolated containing the maltase gene except for the same NHg terminus. pT4-M was digested with BglII/- HindIII and the large 4.1 kb fragment isolated (EFlaA promoter/NH2 terminus and the maltase gene). Both fragments were ligated to each other, yielding pGb-EFMT-3. Sequence analysis confirmed the correctness of all introduced mutations. d) Finally, pGb-EFMT-3 was digested with EcoRV and HindIII to purify a 4.8 kbp. EFlcxA/maltase promoter/gene fragment. From pGb-iA32/G418 (see Figure 6), an approximately 8.3 kb HindIII (partial)/EcoRV fragment was isolated. This consists of the pTZ19R backbone, the ADHI/maltose permease promoter/gene segment and the ADHI/G418<res->segment. Both were ligated and pGb-iRRol was obtained (see Figure 7d). ; 7. pGb- RB2 ; This plasmid serves as a cloning vector to incorporate pieces of DNA into the SIT4 gene of Saccharomyces cerevisiae. The construction includes the following steps (see Figures 8 and 9). ;a) pTZ19R was digested with EcoRI and HindIII to replace the polylinker with a synthetically prepared DNA segment of 40 nucleotides. This piece of DNA was prepared by joining the synthetic oligodeoxynucleotides 3 and 4 (see Figure 8). This short fragment contains EcoRI and HindIII sticky ends. Cloning of this DNA fragment into the pTZ19R vector does not restore the EcoRI and HindIII sites. The synthetic DNA fragment contains at the borders restriction sites for Noti and Sfil and in the middle an EcoRI site as suggested. The resulting plasmid is called pGb-SNENS. b) An EcoRI fragment containing the SIT4 gene (Gottlin-Ninga and Kaback, vide supra) was isolated from pLF24 (also known to have the code pLN420) and cloned into the EcoRI site of pGb-SNENS. This gave pGb-Spons31. Due to the lack of usable reference restraint seats, the orientation is unknown. ;c) Plasmid pGb-Spons 31 contains a unique Bglll site in the middle of the SIT4 gene. This can be used as a site into which any segment of DNA to be transferred to the SIT4 gene by gene replacement can be cloned. To facilitate cloning manipulation, the SIT4 gene is equipped with a synthetic piece of DNA, containing several unique restriction sites. The construction of pGb-RB2 is outlined in Figure 9. The synthetic DNA fragment has two sticky BglII ends. Its orientation in pGb-RB2 as indicated in Figure 9 is based on the restriction enzyme analysis of pGb-RBN3 (see below). ; 8. pGb-RBN3; A 1.9 kb EcoRV/HincII fragment containing the G418<res->gene under the control of the ADHI promoter (see also the construction of pGb-iA32/G418), was cloned into the SmaI site of pGb-RB32 . This gave pGb-RBN3 (Figure 10). ; 9. pGb- BRRO1 ; This plasmid contains the maltose permease gene under the control of the alcohol dehydrogenase I promoter and the maltase gene under the control of the EFlaA promoter. Both are located on an 8.3 kb HindiII fragment cloned into the SIT4 gene. The construction is outlined in Figure 11. To this end, pGb-iRRO1 was digested with HindIII and ligated onto pGb-RB2 x HindIII (treated with calf intestinal phosphatase). This gave pGb—RBRR01. ;10. pGb-RBREGOl ; This plasmid contains the MAL regulator gene under the control of the alcohol dehydrogenase promoter. I. pGb-RBREGOl can be constructed as follows. From p21-40 (R.B. Needleman and C. Michels (1983), supra) the SalI fragment containing the regulatory protein gene was isolated. This fragment is cloned into the SalI site of pTZ18R. This plasmid is commercially available from Pharmacia. The orientation is such that the promoter region of the MAL regulator genes is proximal to the T7 promoter sequence of pTZ18R. Using the sequence primer of pTZ18R and other newly prepared oligonucleotide primers based on the obtained DNA sequence, the promoter area of the MAL regulator gene and the NH2 terminal coding part of the gene can be sequenced. This locates the position of the AUG start codon. When usable restriction sites are absent in the promoter area near the AUG start codon, such a site (for example Bglll) can be introduced into this area via site-directed mutagenesis with an oligonucleotide according to standard methods (see also the construction of recombinant plasmids, part 5b). ;After such mutagenesis the Bglll-SalI fragment (containing the MAL regulatory gene without the promoter region) and an EcoV-BanHI fragment (containing the ADHI promoter, see also Figure 7d, pGb-iRROl), are ligated together into pGb-RB2 ( see Figure 9) so that the MAL regulator gene is under the control of the ADHI promoter. This will give plasmid pGb-iRBREGOl. Sequence analysis using dideoxy chain methods with oligonucleotides on ssDNA as a template can easily confirm the correctness of the cloning steps and the orientation of the promoter. It should be clear that the above-mentioned plasmid constructions only serve as examples to illustrate the invention. Other promoters (preferably Saccharomyces) can be used (or other parts of the same promoter), other integration loci can be chosen, other combinations of maltase, maltose permease and MAL regulator (either under the control of its own promoter or under the control of another, preferably Saccharomyces -promoter), can be produced using one or more integration sites in the yeast genome. It will eventually lead to the optimal ratio between maltase and maltose permease activity which is present throughout the anaerobic fermentation. G. Yeast transformation: Transformation of the yeast strains was carried out according to Ito et al., (H. Ito, Y. Fukuda, K. Murata, A. Kimura (1983), "J. Bacteriology", 153, 163-158). This entails growing Saccharomyces in a standard yeast nutrient medium to a density of 1 to 25, and preferably 4 to 10 OD^q nm. The yeast cells are then harvested, washed and pretreated with chaotropic ions, especially the alkali metal ions, lithium, cesium or rubidium, especially as chloride or sulfate, most preferably the lithium salts at concentrations around 2 mM to 1.0 M and preferably approx. 0.1 M. After incubation of the cells from 5 to 120 minutes and preferably approx. 60 minutes with the chaotropic ion(s), the cells are incubated with DNA for a short time at a moderate temperature and generally from approx. 20 to 35° C for approx. 5 to 60 minutes. Polyethylene glycol is preferably added at a concentration of approx. 25 to 50$, where the total medium can be diluted by adding a corresponding volume of a polyethylene glycol concentrate to thereby achieve the desired final concentration. The polyethylene glycol will be of the 2000 to 8000 dalton type, preferably approx. 4000 to 7000 daltons. Incubation will usually take place within a relatively short time, generally from approx. 5 to 60 minutes. Preferably, the incubation medium is subjected to a heat treatment of from approx. 1 to 10 minutes at approx. 35 to 45°C and preferably approx. 42° C. For selection of transformants, any useful marker can be chosen such as phleomycin (D. Genilloud, M.C. Garrido, F. Moreno (1984) "Gene", 32, 225), hygromycin B (Gritz et al. (1983), "Gene", 25, 178) and aminoglycoside G418 (Jiminez et al., (1980), "Nature", 287, 869). When yeast cells are transformed with integrated plasmids, the integration was directed to the present MAL locus using BglII digested DNA. The integrating plasmids used contained two BglII sites, both in the maltase gene, 1.4 kb apart. This produces double-stranded breaks that are recombinogenic and stimulate the interaction with homologous chromosomal DNA. The gap is repaired from chromosomal information during integration (T.L. Orr-Weaver, J.W. Szostak, R. Rothstein ;(1981), "Proe. Nati. Acad. Sei.", USA 78, 6354). The integration produces one or a few copies of the plasmid vector (J.W. Szostak, R. Cou (1979) "Plasmid", 2, 536). The exact copy number in these transformants, used in CC^ production experiments, has not been determined. ;A scheme was developed to obtain suitable yeast transformants that do not contain heterologous DNA. Examples of heterologous DNA are pTZ19R vector DNA and the selection genes that provide resistance to the antibiotics G418, phleomycin or hygromycin B. In general, the experimental set-up was as follows (figure 12). ;1) One-step gene disruption of a SIT4 gene via transformation of yeast with pGb-RBN3 degraded with Sfil. Transformants were selected for resistance to G418. The yield was strain ApGb-RBN3, in which a SIT4 gene had been replaced with the SIT4 gene interrupted by the G418<r> gene under the control of the ADHI promoter. 2) Strain ApGb-RBN3 was used as a host strain in a co-transformation protocol with pUT332 and with pGb-RBRRO1 degraded with Sfil. pUT332 is a 2>j-derived episomal plasmid containing a gene conferring resistance to phleomycin. The first selection in this cotransformation was carried out on plates containing phleomycin (30 µg/ml). In a certain percentage of these phleomycin1" yeast cells (in the order of 0.1 to 1$) the disrupted SIT4 gene Ø (with pADEI/G418<r>) has been replaced by the co-transformed SIT4 fragment containing altered maltose permease and maltase genes . This second gene replacement resulted in a yeast that is again sensitive to G418. To select yeast cells in which this second gene replacement has occurred, phleomycin^ transformants were replica-plated on plates containing G418 (300 pg/ml). In cells that did not grow the G418<r->gene embedded between SIT4 gene sequences was replaced by the altered MAL genes, embedded between SIT4 gene sequences. 3) Fleomycin1" G418<sens> transformants were then obtained from episomal plasmid pUT332 by cultivation on non-selective medium (ie without phleomycin) for 10-20 generations. The resulting transformant ApGb-pRBRRO1 is sensitive to both phleomycin and G418 and does not contain prokaryotic sequences. All integration is verified at the DNA level by Southern-only assay (data not shown). ;The resulting strain can be used as a host in subsequent transformations using a different integration locus and/or when there are yeast strains that are diploid or polyploid, such as the other allele(s) of the SIT4 gene. Strain A is aneuploid and diploid for the chromosome containing the SIT4 gene. Both SIT4 genes of strain A were used as target sites for gene replacement. This ultimately yielded the transformant ApGb-p2RBRR01 #1. The use of both alleles for integration also most likely increases the genetic stability of the transformant because the second integration removed the polymorphism at this locus. Such polymorphic regions are susceptible to genome rearrangements that can result in the loss of integrated sequences. The transformant thus obtained is analyzed by Southern blot techniques and shows hybridization patterns as predicted from the gene disruption treatments on both SIT4 genes. ;The engineered vector, pGb-RB2, which served as the starting plasmid for pGb-RBN3 and pGb-RBRROl has several useful characteristics, inspired by the following considerations: 1. Often one must integrate a DNA segment constructed by fusing a yeast promoter to a (yeast) coding region of another gene. Gene replacements are of course only achievable if the transformation fragments on both sides have sequences that are homologous to a target sequence in the genome. Therefore, a DNA segment must be nicked at the 3' end of the coding region (see above), which is the derived form from the same gene as the selected promoters. The disadvantage of this method of working is that it requires many cloning manipulations because the same promoter is not always used. In addition, a strong promoter is often chosen, derived from a gene that is functional at the step of interest (during vegetative growth, anaerobic fermentation, and so on). After integration of the engineered fragment, one copy of this gene is rendered non-functional. Therefore, it is desired to direct gene replacement to a locus that is not expressed during vegetative growth. The SIT4 gene (sporulation-induced transcribed sequence) whose expression has been thoroughly studied by Gottlin-Ninja and Kaback (supra) has been chosen, but of course other SIT genes or generally non-coding segments are appropriate. When the DNA construct of interest is cloned into this SIT4 gene, the two resulting halves of the SIT4 gene serve as homologous ends for recombination. One could argue that the chromosomal area where the gene is located is transcriptionally inactive during vegetative growth as a result of a "silencer", analogous to what is described for the HMR locus (A.H. Brand et al. (1985) "Cell" , 4JL, 41-48). This silencer DNA also represses the transcription controlled by promoters, unrelated to adaptation type promoters, and can act on promoters 2600 bp away. Gottlin-Ninfa and Kaback (supra), however, have shown that the EIS3 gene is able to function during vegetative growth if it is integrated into the SIT4 gene. 2. To facilitate cloning, the SIT4 gene is equipped with a synthetic piece of DNA, containing several unique restriction sites (polylinkers with cloning sites). In addition, the polylinker contains stop codons on both sides for translation in all possible reading frames (see Figure 9). This is a safety valve to stop translation of any possible hybrid transcript that may be synthesized across the splice. Such a hybrid transcript may otherwise code for a protein whose function is unknown. 3. To achieve homologous recombination, the DNA segment to be integrated at both ends contains restriction sites for Noti and Sfil, both of which recognize 8 bp sequences. The frequency of the appearance of these restriction sites is very low and therefore it is very unlikely that a DNA segment will be cloned into the polylinker in the SIT4 gene, containing both recognition sites. Thus, once the DNA segment has been cloned into the SIT4 gene on plasmid pGb-RB2, restriction with Noti and Sfil releases a DNA fragment that can recombine by interaction with homologous sequences in the genome, i.e. at the SIT4 locus. Although the ends are part of the Noti or Sfll site and thus not homologous, other studies have shown that, apparently by limited exonuclease digestion in the cell, these sequences are removed (for example H. Rudolph, J. Koenig-Ranseo and A. Hinnen (1985), "Gene", 36 87-95). 4. The commercially available pTZ19r, a pUC19 derivative, is used as starting plasmid. This vector has the advantage that it has a high copy number and that it has an origin of replication of the single-stranded phage etc. This makes it very easy to isolate, after infection with a helper, single-stranded DNA in order to verify the DNA sequence above the junction using primers. This greatly facilitates the detailed description of manipulated DNA. It should be clear that it is possible to apply these improvements not only to baker's yeast, but also to other types of yeast. ;C0£ production measurements ;a) in a synthetic dough medium (sample A). Yeast cells were incubated in YEPMS medium (1% yeast extract; 2% bactopeptone; 3.75% maltose; 1.25% sucrose supplemented with 200 µg/ml G418). Cultivation took place at 30° C until the late log phase. The yeast cells were collected from 6 ml cultures and resuspended in 8.8 ml synthetic dough medium. The composition for this medium was per liter: sucrose 4.6 g; maltose 64.37 g; KH 2 PO 4 2.07 g; MgSO 4 7H 2 O 2.76 g; (NH 4 ) 2 SO 4 0.67 g; casamino acid 2.07 g; citric acid 4.02 g; Na 3 citrate 44.25 g; vitamin B6 9.2 mg; nicotinic acid 46 mg; Ca-D(+)-pantothenate 18.2 mg; biotin 0.23 jjg. During 10 minutes, the suspension was allowed to equilibrate in a water bath at 28°C with moderate stirring, after which the flasks containing the yeast suspension were connected via a tube to a "gas burette". This was filled with a solution that per liter contained 20 ml of indicator solution (1 g methyl red; 0.5 g methylene blue; dissolved in 96$ ethanol), 40 ml IN H 2 SO 4 and a trace of CuS0 4 (dissolved in HN 0 3 ). ;The displacement of the volume of this solution in the district court is a measure of the CC^ formation which is measured during 165 minutes. In each experimental setup, the values for CC^ production are corrected for ambient temperature and pressure to the standard conditions of 28"C and 760 mm Hg. In addition, a correction was made for the amount of yeast. The colorimetric readings at 600 nm for the culture is used as a correction factor to equalize the amount of yeast per C0£ measurement. ;b) in dough (samples B and B'); the CC^ gas formation curves for compressed yeast grown in batches; on molasses were determined in dough without added sugar ( lean dough) (sample B) or with 30% sugar (sample B'). The lean dough was prepared as follows: 1 g compressed yeast (containing 28.5% solids), 34 ml salt solution A (1.25 g NaCl dissolved in 34 ml of distilled water) and 62.5 g of flour were mixed in a Hobart mixer for 30 seconds at speed 1 and 2 minutes at speed 2 to thereby obtain a well-developed dough. ;The dough with 30$ sugar contained 2.0 g of pressed yeast (with 28.5% dry matter), 34 ml salt solution B (0.938 g NaCl in 34 ml distilled water), 62.5 g flour and 1 8.75 g of sucrose (that is, 30$A of sugar in terms of the flour). The mixture was like too lean dough. The dough was then transferred to a round flask. The gas formation measurements started 7.5 minutes after mixing by connecting the flasks containing the dough to a gas burette via a tube (see part a) and were carried out for 165 minutes at 28° C. Where the dry matter content of the pressed yeast differs from the above values, such value used; however, the measured value for CC^ gas-forming ability is corrected by multiplying this by the ratio between required dry matter content and the actual measured value. In each set of experiments, the values for CC 2 formation obtained are corrected for ambient temperature and pressure to 28°C and 760 mm Hg, respectively. In some cases, further calculations have been carried out where the gas values that were obtained have been corrected with a view to the percentage of N in the feed batch's cultured yeast ($ N is an indication of the protein content). Similar percentages of improvement were found without this last correction. ;c) in dough (sample C and C). The CO 2 gas evolution curves for dried yeast such as instant dry yeast prepared according to US-PS 3,843,800 and 4,341,871 were determined in dough with no added sugar (Sample C) or with 30% sugar (Sample C). These tests were carried out in the same way as described in samples B and B', except that 300 mg of dry yeast (containing 96$ dry matter) and 600 mg of dry yeast (containing 96$ dry matter) were used in samples C and C<*>. Before the test the yeast was mixed with the flour and incubated for 10 minutes at 28°C.

Enzymatic analyses

The capacity to transport maltose by yeast cells was determined using [U-^<C>] maltose at a concentration of 15 mM as a substrate at 30° C. Details are published by R. Serrano (supra). Maltase (E.C. 3.2.1.20) was analyzed for the use of p-nitrophenyl-α-D-glucopyranoside as substrate in cell-free extracts. The analysis was carried out according to H. Halvorson and E.L. Elias, "Biochim. Biophys. Acta", (1958), 30, 28.

Substrate consumption and product formation in liquid media The disappearance of maltose and glucose from liquid media was quantified using standard HPLC techniques. One liter of medium contained: 100 g maltose, 10 g glucose, 3.0 g (NH4)2S04, 4.0 g MgS04'7H20, 4 g KH2PO4, 4 g casamino acids ("Difco"), 4 g citric acid'H20, 45 g trisodium citrate•2H20, 10 mg vitamin B1, 10 mg vitamin B6, 40 mg nicotinic acid, 20 mg Ca-D(+)-pantothenate and 0.02 mg biotin. The pH value was adjusted to 5.7. 2 ml medium was added to a suspension of yeast (20 mg dry weight/2.0 ml distilled water). This mixture, called medium A, was incubated at 28°C. Medium B corresponded to medium A, but contained 20 times more glucose. The experiment was carried out under anaerobic conditions.

Protein determination

Protein of cell-free extracts and of whole cells was determined by the microbiuret methods of J. Goa, "Scand. J. Chim. Lab. Invest." (1953), 218. Ovalbumin served as standard.

Quality preservation

Pressed yeast was stored in closed plastic containers at 23" C for 4 days. Quality preservation is defined as the percentage of remaining gas-forming capacity after this period.

Production of pressed yeast

A culture of a yeast strain was grown in a series of fermenters. The cells were grown in 10-liter laboratory fermenters with a net volume of -6 liters. During the fermentation, the pH value and the temperature were kept at the desired value by automatic control. The fermentation recipe used is based on procedures described by G. Butscheck and R. Kautsmann, "Die Hefen", Volume II, "Techonologie der Hefen", pp. 501-591 (1962), Verlag Hans Carl, Nurnberg, FRG and the which is published by G. Reed and H.J. Peppler in "Yeast Technology", AVI Publishing Company Inc., Westport, Connecticut, USA (1973). The cultivation conditions for the final fermentation were special: the molasses used consisted of 80% sugar beet molasses by weight and 20 weight-$ cane molasses, calculated on the basis of 50$

sugar;

the required amount of phosphate was added in the form of mono-ammonium phosphate before inoculation;

the temperature was increased from 28°C to 30°C during the fermentation according to table 1;

nitrogen was added during the fermentation as a 10 µg solution of NH 3 in water according to Table 1;

The pH value was maintained at 5.0 during the first 8 hours of fermentation and then increased according to Table 1 to

6.2 at the end of fermentation;

per kg of molasses containing 50$ of fermentable sugars was

12 mg vitamin Bl added before inoculation.

The yeast obtained by this fermentation was concentrated and washed with tap water in a laboratory nozzle centrifuge. Yeast creams were pressed to a dry matter content of between 26 and 32$.

The obtained protein content ($N x 6.25) varied between 42 and 55$ dry matter as a consequence of different amounts of ammonia added during the fermentation.

Example 1

CO2 production by yeast transformed with 2p-derived plasmids containing genes derived from the MAL6 locus.

Commercial baker's yeast strains A and C were transformed with pGb-eMAL6g (maltose permease and maltase, see Figure 1), pGb-eMAL61 (maltose permease, see Figure 2), and pGb-eMAL63 (MAL regulator, see Figure 3). The MAL genes still contained their original promoters. The nomenclature for these transformed yeast strains is as follows: ApeG418 denotes strain A transformed with peG418. Other transformed strains are indicated analogously. The effects of these plasmids on CC>2 production in synthetic dough medium are summarized in Table 2. The host strain, transformed with parent peG418 (see Figure 1) serves as a reference because the mere presence of a multicopy plasmid has been found to have a negative effect on the gas formation.

All transformants showed significant CC^ formation enhancements relative to the control. The combination of extra copies of both maltose permease and maltase genes gives the best improvement, approx. 40$ in tribe A and 18$ in tribe B.

The transformants ApGb-eMAL6g and CpGb-eMAL6g have also been tested in dough without added sugar. In this case, the yeast was grown in batches on molasses.

Here, too, significant improvements were achieved in (^-production) (table 3).

Table 2

Gas production of strain A and strain C, transformed by 2jj-derived MAL plasmids, relative to vector-transformed strains, (^-production was measured in synthetic dough medium (see experimental procedure) and corrected to 285 mg dry matter. The data shown are means of several attempt.

Table 3

Relative gas formation in dough of strains A and C, transformed with 2p-derived plasmids peG418 and pGb-eMAL6g, COg production being corrected to 285 mg dry matter.

Example 2

CO^ production by yeast strains transformed with integrating plasmids containing recombinant maltase and/or maltose permease genes.

Parental yeast strain A was transformed with pGb-iA32/G418 (main features: ADHI/maltose permease; see Figure 6) and pGb-iRRol (main features: ADHI/maltose permease and EflaA/maltase; see Figure 7).

Parental strain Å and both integrative transformants are grown in batches on molasses corresponding to the commercial aerobic fermentation (see table 1). After harvesting the cells, the CO2 production was measured in a standard dough sample without added sugar. The gas production, analyzed in this sample, is summarized in table 4. The integration of pGb-iA32/G418 into the chromosome of strain A significantly improves gas production in the dough. The relative improvement varies somewhat depending on the measurement time (see table 4). When, in addition to an altered maltose permease gene, an altered maltase gene is integrated into the chromosome of commercial strain A using pGb-iRRol, the gas-forming ability is further improved. In this typical experiment, approx. $30 more CO2 produced after 165 minutes in a lean dough, which corresponds to a level of approx. 410 ml CO2/285 mg dry weight of yeast.

In a 30$ dose of sugar paste, no significant differences were noted in the CC^ production of the transformants compared to the parent strain (approx. 190 ml CO2/285 mg dry weight <g>yer).

The achieved improvement in heaving activity is maintained during storage at 23°C. The loss of heaving activity during storage is virtually identical for parent strain A and the new strains (see Table 5).

Table 4

Relative gas formation for strain A and its rDNA derivatives equipped with altered maltase and/or maltose permease genes. The gas values have been corrected to 285 mg dry matter. No sugar was added.

Table 5

Quality conservation of strain A and its rDNA derivatives equipped with altered maltase and/or maltose permease genes. The leavening activity was measured as in table 4 after the press yeast had been kept at 23° C. for 4 days.

Example 3

CC"2 production for a yeast strain containing recombinant maltase and maltose permease genes and no heterologous DNA.

Parent strain A was genetically modified so that a pADHI/- maltose permease gene and a pEFlaA/maltase gene were introduced into the SIT4 gene on both homologous chromosomes. This strain, abbreviated as ApGb-p2RBRR01 #1, was constructed using the methods and plasmids described previously (see the sections on transformation and construction of the plasmid of pGb-RBN3 (Figure 10) and pGb-RBRRO1 (Figure 11 ) and the general scheme for transformation via gene replacement. Parent strain A and the homologous transformant ApGb-p2RBRR01 #1 are grown in batches on molasses corresponding to the commercial aerobic fermentation (see Table 1). After harvesting the cells, the C02 production is measured in a standard dough sample without added sugar.

Table 6 summarizes the results. In shortcrust pastry, the improvement is approx. 18$ after 165 minutes, which corresponds to a level of approx. 367 ml C02/285 mg dry weight yeast. This strain contains two copies each of a maltose permease gene under the control of the ADHI promoter and a maltase gene under the control of the EflocA promoter. In table 4 it is shown that the strain ApGb-iRROl has an improvement of approx. 30$. An initial estimation of the number of pGb-iRROl molecules integrated into a MAL locus in strain A gives a copy number of integrated plasmid molecules of at least 3. By further increasing the copy number of altered maltose permease and maltase genes in strain ApGb-p2RBRR01 #1 (for for example by integration into other sporulation-specific genes), a homologously transformed yeast strain can be obtained with at least similar gas formation levels as strain ApGb-iRRO1.

Table 6

Relative gas formation for strain A and its homologous rDNA derivatives equipped with altered maltase and maltose permease genes. The gas values have been corrected to 285 mg dry matter. No sugar was added to the dough.

Example 4

Enzyme activities and substrate uptake rates of yeast strains transformed with integrating plasmids containing recombinant maltase and/or maltose permease genes.

As described above, the expression of maltose permease and maltase is subject to maltose induction and glucose repression. This phenomenon is shown in Figure 13 for wild-type cells of strain A. Specific activities of maltose permease and maltase do not increase until most of the glucose is consumed.

The activity of maltose permease at the start of the dough rise is increased by introducing an altered maltose permease gene into the yeast genome (strain ApGb-lA32/G418) as shown in figure 14. Surprisingly, maltase activities were also increased in this construct (figure 15). This new strain ApGb-iA32/G418 fermented maltose faster than the parent strain A in medium A containing maltose as the main carbon and energy source (Figure 16). In medium B containing glucose as the main carbon and energy source, this effect was less pronounced (figure 17).

In addition to an altered maltose permease gene, an altered maltase gene was integrated into the chromosome giving the strain ApGb-iRROl. This strain fermented maltose at even higher rates in medium A (Figure 16) and also in medium B (Figure 17). Despite the high extracellular concentration of glucose, significant amounts of maltose were metabolized by this new strain. Strain ApGb-iRRol showed even higher specific activities for maltase and maltose permease during the dough rise than parent strain A and strain ApGb-iA32/G418 (Figures 14 and 15).

Claims (19)

1. Transformed yeast of the genus Saccharomyces preferably Saccharomyces cerevisiae, characterized in that it is capable of improved production of carbon dioxide compared to a non-transformed origin of the yeast by fermentation in a medium comprising maltose, where the maltose is fermentable both by the origin and the transformed yeast, and that the transformed yeast comprises a DNA construct comprising at least one gene capable of expression in the transformed yeast which encodes a maltose permease, maltase or a maltose regulatory protein. 2. Yeast according to claim 1, characterized in that the genes or combinations thereof are brought under transcriptional control that is not sensitive to glucose repression and is not a victim of maltose induction. 3. Yeast according to claim 1, characterized in that the gene is under the transcriptional control of alcohol dehydrogenase I (ADHI) and/or translation elongation factor (EFlocA) promoter. 4. Yeast according to any one of claims 1 to 3, characterized in that the construct is part of an episomal element. 5. Yeast according to any one of the preceding claims, characterized in that the construct is integrated into a chromosome in the yeast. 6. Yeast according to any one of the preceding claims, characterized in that it comprises at least two of the said constructs. 7. Yeast according to any one of the preceding claims, characterized in that the yeast is free of heterologous DNA. 8. Yeast with a moisture content of from 3 to 8%, characterized in that it is obtained by drying a yeast obtained according to any one of claims 1 to 7. 9. Yeast, characterized in that it is obtained by strain improvement procedures other than DNA-mediated transformation, using a yeast according to any one of claims 1 to 14. 10. Yeast according to claim 1, characterized in that it is selected from Saccharomyces cerevisiae strain ApGb-iA32/G418, Saccharomyces cerevisiae strain ApGb-iRRol, Saccharomyces cerevisiae strain ApGb-eMAL6g, Saccharomyces cerevisiae strain ApGb-eMAL61, Saccharomyces cerevisiae strain ApGb-eMAL63, Saccharomyces cerevisiae strain ApGb-eMAL6g and Saccharomyces cerevisiae strain ApGb-eMAL61. Saccharomyces cerevisiae strain ApGb-p2RBRR01 #1. 11. Pressed, instant dry or active dry yeast, characterized in that it is obtained from a yeast according to any one of claims 1 to 10. 12. Pressed yeast according to claim 11, characterized in that it shows a gas production of at least 340 ml/285 mg dry weight of yeast during 165 minutes in sample B and a gas production of at least 170 ml/285 mg dry weight of yeast during 165 minutes in sample B' , and that it preferably shows a gas formation of 380-450 ml/285 mg dry weight of yeast in sample B during 165 minutes and a gas production of 180-240 ml/285 mg dry weight of yeast during 165 minutes in sample B', all preferably a gas production of at least 400 ml/285 mg dry weight yeast in sample B and respectively 190 ml/285 mg dry weight yeast in sample B'. 13. Pressed yeast according to claim 12, characterized in that it shows a gas production of 400-500 ml/285 mg dry weight of yeast during 165 minutes in sample B, and preferably a gas production of 440 1/285 mg dry weight of yeast during 165 minutes in sample B. 14. Dry yeast or an active dry yeast according to claim 11, characterized in that it has been obtained by drying the pressed yeast according to claims 12 or 13. 15 . Dry yeast according to claim 11, characterized in that it shows a gas production of 310-360 ml/285 mg dry weight of yeast during 165 minutes in sample C and a gas production of 145-195 ml/285 mg dry weight of yeast during 165 minutes in sample C, preferably a gas production of at least 330 ml/285 mg dry weight of yeast during 165 minutes in sample C or shows a gas production of at least 155 ml/285 mg dry weight of yeast during 165 minutes in sample C, or which shows a gas production of 320-400 ml/285 mg dry weight of yeast during 165 minutes in sample C, preferably shows a gas production of at least 350 ml/285 mg dry weight of yeast during 165 minutes in sample C. 16. Use of a yeast according to claim 1 for the production of dough or similar products. 17. Use of a yeast according to claim 1 for the production of alcoholic beverages and other alcoholic products. 18. Use of a yeast according to claim 1 for the production of bread and related products. 19. Use of a yeast according to claim 1 for the production of an enzyme with maltase or maltose permease activity.