FI100473B - Accelerated fermentation of sugars is brought about by new yeast strains, the method of their preparation and their use - 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

100473

Accelerated fermentation of sugars is achieved by new strains of yeast, the method of their preparation and their use.

The invention relates to novel yeasts capable of improving the fermentation of sugars, to a process for the construction of such yeasts and to the use of these developed yeasts.

It is already known, for example, that yeast strains belonging to the genus Saccharomyces are capable of fermenting sugars to approximately equimolar amounts of carbon dioxide and ethanol under anaerobic conditions. The ability of yeast to raise the dough is due to this fermentation. The baking yeast available as a commercial product is available in several formulations, i.e., yeast compress, which is fresh yeast, and dried yeast. Dry yeast is available as active dry yeast and quick dry yeast with a moisture content of 6-8% and 3-6%, respectively.

One of the first steps in sugar metabolism in yeast is the transport of sugar molecules across the cell membrane, or plasma membrane. Yeast expresses specific carriers for different sugars. For example, maltose uptake depends on the specific maltose permease. This carrier may exist in two forms that differ in terms of maximum rate (Vmax) and affinity constant (Km) (A. Busturia and R. Lagunas, Biochim. Biophys. Acta 820, 324 (1985)). The migration of maltose through the yeast cell membrane is related to the electrochemical proton gradient of this membrane. One proton is symbolized for each molecule of maltose taken up (R. Serrano, Eur. J. Biochem. JM, 97 (1977)).

Within the cell, maltose is hydrolyzed to two 100473 2 glucose molecules in a maltase (alpha-glucosidase) catalyzed reaction. Glucose is then converted to carbon dioxide and ethanol in the Embden-Meyerhof metabolic pathway. Compared to glucose fermentation, maltose fermentation requires two additional enzymes, maltose permease and maltase. Maltose induces and the synthesis of these enzymes is repressed by glucose, fructose or mannose. In doughs to which no sugar has been added ("lean doughs"), maltose is the sugar most available for yeast use. If sucrose is added to the dough, this is hydrolyzed extracellularly by the yeast to glucose and fructose. Yeast then takes up these hexoses by the action of certain permeases.

In general, the addition of sucrose to maltose-containing media, such as dough, has been found to inhibit maltose metabolism in yeast cells. This is because glucose represses the transcription of genes encoding maltose permease and maltase (RB Needleman, DB Kaback, RA Dubin, EL Perkins, NG Rosenberg, KA Sutherland, DB Forrest, and CA Michels, Proc. Natl. Acad. Sci USA et al. 1, 2811 (1984)).

The genes required for maltose uptake and hydrolysis are clustered at the MAL locus. · (R.B. Needleman et al., Supra). Saccharomyces strains may contain up to five MAL loci (MAL 1-4 and MAL6) that are not related to each other located in telomeres on different chromosomes (J.L. Celenza and M. Carlson, Genetics 109, 661-664 (1985)). The MAL locus contains genes encoding maltose permease, maltase, and one or more regulatory proteins (MAL regulators) required for the induction of maltose (RB Needleman et al., Supra; JD Cohen, MJ Goldenthal, T. Chow, B. Buchferer, and J. Marmur, Mol. Gen. Genet. 200, 1 (1985); RA Dubin, EL Perkins, RB Needleman and CA Michels, Mol. Cell. Biol. 6, 2757 (1986)). Said genes 100473 3 have been isolated and cloned (AOJD Cohen et al., Supra; RB Needleman et al., Supra; HJ Federoff, JD Cohen, TR Eccleshall, RB Needleman, BA Buchferer, J. Gia-calone and J. Marmur, J. Bacteriol. 149, 1064 (1982)).

As mentioned above, the fermentation of yeast in lean dough depends on maltose as the main substrate. Maltose is formed in the dough from starch by the action of amylases, which are normally present in flour. In addition, the flour contains varying amounts (0-0.5%) of free sugars such as glucose, raffinose, etc. (H. Suomalainen, J. Dettwiler and E. Sinda, Process Biochem 7, 16 (1972)). Yeast consumes these sugars quickly. Several studies have been published on the relationship between baker's yeast maltose fermentation and raising activity. In some cases, a positive relationship was found between the fermentation rate of maltase and the activities of Maltase and maltose permease. In contrast, no positive association could be observed between maltase and maltose permease activities and lean dough lifting ability (P. Hautera and T. Lövgren, J. Inst. Brew. 81, 309 (1975); T. Lövgren and P. Hautera, Eur.

J. Appl. Microbiol. 4, 37 (1977)).

When yeast cells were transformed with multicopy plasmids containing the genes encoding maltase and maltose permease, a quadrupling of the specific activity of maltase was achieved, but no increase in maltose permease activity. The introduction of additional genes encoding a regulatory protein resulted in a moderate increase in maltase specific activity, but even now no change in maltose permease activity could be observed (J.D.

• Cohen et al., Supra). These investigators did not measure the carbon dioxide and ethanol production of the resulting transformants. In fact, prior art knowledge did not encourage such tests, as it had been repeatedly shown that the activities of maltose permease and maltase had nothing to do with carbon dioxide production (raising activity) in lean dough (H. Suomalainen, J. Dettwiler, and E. Sinda, supra; H Suomalainen, Eur. J. Appl. Microbiol. 1, 1 (1975); P. Hautera and T. Lövgren, supra; T. Lövgren and P. Hautera, supra).

The present inventors have now found that yeasts transformed with an integrating plasmid, exemplified below, give higher levels of maltose permease and maltase activity than the untransformed strain. These higher maltase and maltose permease activities surprisingly coincide with carbon dioxide production or uptake capacity, which was also observed in the case where the yeast was transformed with episomal vectors.

These yeasts developed show higher metabolic rates, resulting in, for example, higher production of carbon dioxide and ethanol in media containing sugars, such as maltose, as the main source of carbon and energy. These are genetic engineering methods used in such a way that the rate of fermentation of maltose in said yeasts increases really strongly, regardless of the presence of other sugars, such as glucose.

In addition, these developed yeast strains have excellent dough-raising ability (gas production). Correspondingly, the acceleration of ethanol production shown by these yeasts results in a shorter fermentation time of the yeast used and a higher amount of alcohol produced in the production of industrial alcohol for a certain period of time.

In addition, if maltose (or its accumulation) inhibits: the activity of enzymes that convert sugar or starch, rapid removal of maltose is advantageous in that fermentation process. Using the method of the present invention, a yeast can be provided with the additional gene (s) encoding the maltase (s) and / or maltose permease.

The present invention provides a transformed yeast and a method of obtaining said yeast having a higher sugar fermentation rate, said method comprising introducing into the yeast at least one, preferably homologous, DNA construct containing at least one gene encoding a protein that promotes uptake and / or its initial metabolic alteration, and said yeast is capable of expressing said gene.

The rate of sugar fermentation can be increased in several stages of raising; the rate can be increased by, for example, the fermentation of maltose or sucrose.

By homologous DNA is meant DNA from the same yeast family. For example, Saccharomyces is transformed with DNA derived from Saccharomyces. In this way, it is possible to improve properties already present in the yeast family without introducing new properties that are not already present in the family. An increase in the fermentation rate of sugars can be achieved under aeorobic and / or anaerobic conditions. Genes of interest include e.g. permeases, especially maltose permease, saccharidases, especially maltase, kinases, especially hexokinases and glucokinase, etc. The present invention can be applied, for example, to a carrier protein required for glucose or fructose uptake and from .

The present invention also provides efficient methods for transferring at least one homologous DNA construct to yeast.

Preferably, the invention is applicable to a construct comprising at least one gene encoding a protein that promotes maltose uptake and the initial stage of maltose glucose metabolism.

100473 6 In this way, yeasts can be obtained which have several advantages over the original strains (host strains). The advantages of such a developed yeast can be seen in particular in the increased production of ethanol and carbon dioxide. For example, when this invention is applied to baking yeast, the baker achieves a huge advantage because now he needs less time or less yeast to raise the lean dough due to the higher raising activity of the new strains.

It will be appreciated that the transformed yeast of the invention may be used as a starting strain in strain development operations other than DNA-mediated transformation, for example in protoplast fusion, mass pairing and mutation. The strains thus obtained are considered to be part of the present invention.

According to a preferred embodiment of the invention, the new strains consume considerable amounts of maltose in the presence of glucose. Thus, bakers may save on sugar costs because less sugar needs to be added to make sweet doughs.

It is already well known that the performance (lifting ability) of osmotolerant yeasts * is poor in lean doughs. Osmotolerant yeasts refer to yeasts that have good performance in sweet doughs. For example, doughs for sweet bakery products contain 10 to 30% sugar (based on the weight of the flour). Thus, when an osmotolerant strain is selected as the host to be transformed according to the invention, an osmotolerant yeast is obtained which can be used not only in sweet doughs but also in lean doughs because of its ability to ferment maltose according to the invention. Thus, the baker only needs to use one type of yeast for both sweet and lean doughs.

The need for yeast strains with good performance in both lean and sweet doughs is described in, for example, EP-A-128524 and DE-A-2757778. In order to obtain yeast strains with good performance in sweet and lean dough, EP-A-128525 describes a protoplast fusion method and DE-A-2757778 describes a selection method for obtaining such yeast strains by hybridization or selection of strains by hybridization. Both of these methods require a lot of experimentation and the results are unpredictable and non-reproducible. Using the method of the present invention, controlled and reproducible results can be obtained in the transformation of yeast strains. Testing of the yeasts produced in accordance with the present invention can be kept to a minimum because the properties of the strain itself do not change substantially, only the improved properties are altered properties, as described.

The present invention provides compressed yeast with a gas production of at least 340 ml / 285 mg dry weight yeast in 165 minutes in Experiment B and at least 170 ml / 285 mg dry weight yeast in Experiment B '. Experiments B and B 'are described below. Preferably, the compressed yeast gas production is 380-450 ml / 285 mg dry weight yeast in 165 minutes in Experiment B and 180-240 ml / 285 mg dry weight yeast in Experiment B ', and more preferably at least 400 ml / 285 mg dry weight yeast in Experiment B and at least 190 ml / 285 mg dry weight yeast in experiment B '. From this compressed yeast, it is preferable to produce quick-drying yeast or active dry yeast. When the compressed yeast is dried, 15-25% of the raising activity, based on the dry matter, is generally lost. The present invention also provides dried yeast (moisture content 3 to 8% by weight) having a gas production of 8 100473 310 to 360 ml / 285 mg dry weight yeast in 165 minutes in Experiment C and 145-195 ml / 285 mg dry weight yeast in Experiment C * and preferably at least 330 ml / 285 mg dry weight yeast in test c and at least 155 ml / 285 mg dry weight yeast in test C. The gas production values measured for the yeast according to the invention have never been found even in commercial strains when tested in experiments C and C. The invention can also be applied to yeasts which have a good lifting activity behavior in sugar doughs in the range of 0-6 or 0-10%. Thus, it is possible to produce compressed yeasts with a gas production of 400 to 500 ml / 285 mg dry weight yeast in Experiment B, but preferably the gas production of these compressed yeasts is at least 440 ml / 285 mg dry weight yeast. Dried yeasts with a gas production of 320 to 400 ml / 285 mg dry weight yeast can then be obtained in 165 minutes in Experiment C, but preferably the dried yeast gas production is at least 350 ml / 285 mg dry weight yeast.

These new strains show the same advantages in fermentations, ie fermentations producing alcohol for human consumption or for industrial use.

· As the overall metabolic rate of these new strains has increased when maltose acts as a substrate, the overall rate of production of metabolites such as glycerol and aroma compounds will increase equally.

The present invention also provides vectors comprising a DNA construct encoding one or more proteins involved in maltose fermentation. The present invention further provides a microbial host, preferably a yeast, representing, for example, a Saccharomyces species, transformed with a vector of the invention. These vectors may be self-

• I

s id; jlll I i I II

100473 9 replicable and preferably contain a gene or gene combination such that the gene or genes are selected from maltose permease, maltase and a maltose regulatory protein. It has been found above that yeast transformed with such a vector shows an increased malting capacity of maltose, resulting in increased carbon dioxide production in the dough. However, these additional genes are located in episomes and it is known from the literature that such extrachromosomal molecules are easily lost in non-selective culture (i.e. in this particular case without G418) (CD Hollen-Berg (1982), Gene Cloning 12, Organisms other than E. coli , edited by PH Hofschneider, W. Goebel, Springer Verlag, 119, SA Parent, CM Fenimone and KA Bostian (1985),

Yeast 1, 83). From a practical point of view, it is advantageous to grow the yeast non-selectively and thus to construct a number of integrating plasmids which contain, preferably modified, the maltase gene and / or the maltose permease gene. By "modified" is meant that the native promoter has been replaced with another, preferably homologous, promoter. If methods are developed that allow stable proliferation of plasmids without selection pressure, integration of the newly introduced DNA will no longer be a prerequisite for stable transformants. acquiring.

It is known from the literature that one cell contains 20 to 100 such additional plasmid molecules (CD Hollenberg (1982), supra; A. Takagi, EN Chun, C. -Borchird, S. Harashima and Y. Oshima, Appl. Microbiol. Biotecnol. 23, 123; J. Mellor, MJ Dobson, NA Roberts, NJ Kingsman, and SM Kingsman (1985) Gene 22/215).

The expression level of episomal genes can be further increased when the original promoters are replaced with stronger ones. It seems obvious that in the future stable replication of plasmids is possible in the absence of 100473 10 selection pressure.

According to the invention, it is preferred to integrate the maltase gene and / or the maltose permease gene into the yeast chromosome by transformation with linear plasmids (see T.L. Orr-Weaver, J.W. Szostak, R. Rothstein (1981) Proc. Natl. Acad. Sci. USA 78, 6354). The resulting yeasts are stable transformants, i.e. the modified maltase gene and / or maltose permease gene remain inherited even without selection pressure.

It is already known that during integration, only one or a few gene copies located in the plasmid integrate into the chromosome. Thus, in order to achieve similar increases in carbon dioxide production as when using episomal vectors, the level of gene expression can be advantageously altered by the use of strong constitutive promoters that are not sensitive to glucose repression.

Such promoters are prioritized to compensate for the difference in the number of gene copies between yeasts transformed with episomal and integrative vectors and to prevent the effects of glucose repression. For example, it has been found that when fermented in a medium in which maltose is the main carbon and energy source and which contains glucose at a relatively low concentration, the mRNA of maltose permease and maltase decreases to barely detectable levels during the first 30 to 40 minutes. When glucose is depleted, the induction of gene expression by maltase results in a rapid increase in the amount of both mRNA grades.

As already mentioned above, the genes can be used with a native, i.e. wild-type promoter, or the promoter can be replaced by a different promoter, preferably homologous. In particular, if the wild-type promoter is controllable or inducible, it may be advantageous to use constitutive transcription or a stronger or weaker promoter. Conversely, when the wild-type promoter is constitutive, it may be advantageous to use a regulated or inducible promoter or a stronger or weaker promoter.

It is advantageous to use strong promoters, especially if the copy number of the structure in the host is relatively small. Strong promoters are generally those involved in the production of proteins produced in large amounts during the yeast growth cycle, or, in the case of regulated promoters, they function at some desired stage of the yeast growth cycle associated with the present invention.

Of particular interest are promoters associated with the glycolytic cycle of yeast, and include e.g. alcohol dehydrogenases I and II, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase, triisophosphate isomerase, glyceraldehyde diphosphate dehydrogenase, phosphoglycerate kinase, enolase, phosphoglyceromodotase, pyruvate kinase Other promoters that are associated with proteins produced in large quantities include e.g. promoters associated with ribosomal expression, such as promoters for transcription initiation factors, extension factors, and the like. Continuing factors. mention should be made e.g. EF-1 and EF-2, etc.

Of particular interest is the use of promoters in connection with structural genes involved in maltose metabolism, notably maltase, maltose permease and the MAL regulator. Such promoters may be constitutive or regulatory, as long as the promoter is induced during the fermentation of the sugar. For example, many glycolytic promoters are activated in the presence of sugar or some sugar metabolism product, such as ethanol. Thus, for example, the promoter of alcohol dehydrogenase 100473 12 is active when yeast raises the dough. Similarly, promoters involved in cell proliferation are active during dough raising. In addition, if a promoter is provided that is not under the control of the MAL regulator, yeast can be used in the presence of glucose without repression. These genes also do not require maltose to induce.

If a wild-type promoter is used with the desired structural gene, it may be advantageous to achieve higher production of the regulatory protein. In this way, a higher amount of regulatory protein can be maintained when the inducer is present. For example, when maltose is present, the MAL regulatory protein is expressed in large amounts to allow expression and regulation of other proteins involved in maltose metabolism in the regulation of the MAL regulatory protein.

To alter gene expression, as described in detail in the experimental section, the original promoters and unreadable leader sequences (or portions thereof) are replaced with those of alcohol dehydrogenase I (ADHI) and reading extension factor EFαA, preferably derived from a host yeast, e.g. Saccharomycin. as a result, expression becomes insensitive to glucose-induced repression and induction of expression also becomes independent of maltose.

It has been found that yeasts transformed with these integrating plasmids show higher maltase and maltose permease activity than the untransformed strain. These elevated maltase and maltose permease activities surprisingly coincide with carbon dioxide production, i.e., ascending activity, as was also observed for yeasts transformed with episomal vectors.

The improved lifting capacity thus obtained is also maintained during storage of the 100473 13, even at high temperatures, for example at 20-25 ° C. The relative loss of lifting activity during storage is substantially identical to the base strains and the strains according to the invention. The changes obtained do not affect the uptake capacity of the sweet doughs, as the uptake capacity of the new strains transformed with the integrating plasmids is as good as that of the host strain.

The yeast host in question will have at least one copy of the structure, possibly two or more, but usually no more than about 200, depending on whether the gene integrates inheritance, amplifies, or is present as an extrachromosomal element with multiple copies. Integration or non-integration can be selected depending on what stability is required to maintain the chromosomal extracellular element produced, what copy number is desired, what level of transcription is desired, achieved depending on the number of copies, and so on.

The construct may contain one or more structural genes having the same or different promoters. The construct may be conventionally prepared by isolating the desired genes from a suitable host or by synthesizing all or part of the genes or by using combinations of these methods. Accordingly, regulatory signals and transcription and reading start and stop regions can be isolated from a natural source or synthesized or made available by combining these methods. The various fragments can be subjected to endonuclease cleavage (restriction), annealing, sequencing, in vitro mutation, primer repair, etc. These various transformation procedures are well known in the literature and are used for certain purposes.

The various fragments can be combined, cloned, isolated and sequenced by conventional methods. After each transformation procedure, a DNA fragment or combination of fragments can be inserted into a cloning vector, transformed into a cloning host, e.g., an E. coli, vector, grown into a cloning host, lysed, isolated from a plasmid, and analyzed by restriction analysis, sequencing, or any combination thereof.

Various vectors can be used to develop the structure and transform the host cell. Such vectors may be e.g. cloning vectors, expression vectors and vectors providing host integration, or DNA alone can be used for transformation and integration.

The cloning vector is mainly characterized by the fact that it contains an origin of replication in the cloning host, a check mark on the basis of which the host containing the cloning vector can be selected, perhaps one or more multi-linkers or additional sequences for insertion, selection, modification, facilitation of sequencing. In addition, shuttle vectors may be used, in which case the vector may have two or more origins of replication which allow the vector to replicate in more than one host, for example a prokaryotic host and a eukaryotic host.

Expression vectors may typically include a construct that includes start and stop regions for transcription and reading, or may lack one or both regulatory regions that are then included in the expression vector when the sequence encoding the protein product is inserted. Thus, the construct can be placed in a gene with functional transcriptional and reading regions, with the placement occurring near the 5 'end, or the existing gene and construct will be under the control of existing regulatory regions. Normally, it would be desirable for the start codon to be the 5 'end of an existing start codon, unless the fusion product is acceptable or the start codon is not in reading phase with the existing start codon.

100473 15

In other cases, expression vectors are present that have one or more restriction sites between the start and stop regulatory regions so that the structural gene can be placed at the restriction site (s) to regulate these regulatory regions. Of particular interest in the present invention are expression vector Reina, either in extrachromosomal stable maintenance or integration, constructs and vectors that are free of heterologous (non-Saccharomyces) DNA in their stable form in the host.

Furthermore, the present invention provides methods for providing yeasts that have all of the advantages described above but have eliminated prokaryotic DNA sequences. This has been accomplished by gene replacement technology (R. J. Rothstein (1983) Methods in Enzymology, 101, 202). In accordance with the present invention, these techniques have now been advantageously applied to Saccharo-m ^ ces cells. For example, Saccharomyces cells have been transformed with a vector containing genes encoding modified maltase and / or maltose permease located in a gene specific for Saccharomyces sporulation (E. Gottlin-Ninga, DB Kaback (1986), Mol. Cell. Biol. 6, 2185). . Once this DNA has been introduced into a Saccha-romyces host cell, homologous recombination of the newly imported DNA with a chromosomal spore-forming gene occurs. In this case, the modified maltase and / or maltose permease genes, which are placed between sequences specific for sporulation, integrate into the chromosome. The transformants thus formed are completely devoid of prokaryotic DNA.

• It is clear that even better results can be obtained if the optimal ratio of maltase to maltose permease activities is determined. This can be accomplished by altering the promoter of both genes or by integrating the (modified) maltase and and maltase permease genes in different amounts into the yeast genome, e.g., using multiple integration loci, using the methods described below.

An optimal ratio of maltase to maltose permease activities can also be achieved by using the maltase and maltose permease genes that encode the maltase and maltose permease isoenzymes. In addition, any enzyme having at least maltase or maltose permease activity can be used.

In addition, the genes encoding the MAL regulatory protein can be integrated into the genome in a similar manner (see also Example 1), either under the control of its own native promoter or under the control of another promoter, preferably of gpnrharomyces. This may also be useful to achieve an optimal ratio of maltase to maltose permease activities.

List of deposited stocks.

The following stocks are deposited in the collections of the Centraal Bureau voor Schimmelcultures, located in Baarn, the Netherlands:

Saccharomyces cerevisiae 237 Ng (strain A), deposited with CBS Collections No. 158.86 on March 25, 1986;

Saccharomyces cerevisiae DS 15543 (strain C), deposited in the CBS collections under number 406.87 on! Of 3 September 1987

Escherichia coli with plasmid p21-40, deposited with CBS Collections No. 400.87 on August 28, 1987;

Escherichia coli with plasmid pYEF46, deposited with CBS Collections No. 401.87 on August 28, 1987;

Escherichia coli with plasmid pY6, deposited with CBS Collections No. 402.87 on August 28, 1987;

Escherichia coli with plasmid peG418 was deposited in CBS collections at 160.86 on 25.

17 March 100473, 1986;

Escherichia coli with plasmid pTZ19R, deposited with CBS Collections No. 405.87, dated September 3, 1987;

Escherichia coli with plasmid pTZ19R / ADHI, deposited with CBS Collections 404.87 on September 3, 1987;

Escherichia coli with plasmid p153-215 AK, deposited with CBS size number 403.87 on September 3, 1987;

Escherichia coli with plasmid pLF24, deposited with CBS size number 156.88 on March 8, 1988;

Escherichia coli with plasmid pUT332, deposited with CBS size number 158.88 on March 8, 1988;

Escherichia coli with plasmid pTZ18R was deposited in CBS Collections No. 480.88 on July 27, 1988.

Brief description of the pictures:

Figure 1 shows the construction of plasmid pGb-eMAL6g. The arrows indicate the direction of transcription of said genes. Plasmids are plotted schematically, not to scale. Abbreviations: G418, Tn5 gene (under the control of the ADHI promoter) conferring resistance to G418; P = PvuI; X = XbaI; S = Sali; H = HindIII; CIP = calf visceral phosphatase.

Figure 2 shows the construction of plasmid pGb-eMAL61. Plasmids are plotted schematically, not to scale. Abbreviations: Δ maltase gene with partial deletion; B = BglII. See. also the description of Figure 1.

Figure 3 shows the construction of plasmid pGb-eMAL63. Plasmids are plotted schematically, not to scale. Abbreviations: (K) = filled Kpnl site; (S) = filled Hall section; Klenow = large DNA subunit; H = HindIII. See also the description in Figure 1 100 100473.

Figure 4 shows the construction of plasmid pGb-M6g (A-9). Abbreviations: H = HindIII; St = Stul, Klenow = DNA

a large fragment of polymerase I; EV = EcoRV; Xh = XhoI; M = MluI; fl ori = origin of replication of phage f1; amp = ampicillin resistance gene; s.s. = sequencing primer. The arrows indicate the 5 'to ^ * 3' direction. The deleted area is marked with dashed lines and 4: 11a.

Figure 5 shows the sequence of plasmid pGb-M6g (A-9).

(a) Maltase and maltose permease genes. The arrow indicates the direction of transcription. St (Stul) served as a starting point for the construction of the deletion mutant. Below this map are the essential parts of the intergenic region.

b) Sequence of plasmid pGb-M 6g (Λ-9). The deleted range extends from position -9 to position -417. Multi-linker refers to oligonucleotides fused to Bal31-treated DNA (see also Figure 4).

Figure 6 shows the construction of plasmid pGb-iA32 / G418. Arrows indicate the direction of transcription. Plasmids are plotted schematically and are not to scale. Abbreviations: H = HindIII; EV = EcoRV; fl ori = origin of replication of phage f1; amp = ampicillin resistance gene; G418, Tn5 gene (ADHI promoter) ’! conferring resistance to G418; S = Smal; He =

HttncII; pADHI = alcohol dehydrogenase I gene promoter + 5 'portion of the leader sequence (dashed region).

Figure 7 shows the construction of plasmid pGb-iRRO1.

a) Plasmid pT4 is not drawn to scale. Abbreviations: E = EcoRI; B / Bg = BamHI / BglII junction; H = HindIII.

b) Mutation of plasmid pT4 to link the EF1 «A promoter + 5 'leader sequence to the five amino acid codons of the N-terminus of the maltase gene so as to create a BglII site. The essential sequences are shown.

The mutation primer is partially complementary:! 9il! UH I t I il 100473 19 (dotted) with the EF1'X.A sequence. In the region of the mismatches are the maltase codons and, when ringed, the BglII recognition site (Note that the direction shown in the mutation primer is 3 '- * - 5', i.e. the BglII site should be read from right to left (5 '- ^ 3').

- The N-terminal five-amino acid codon is labeled in the maltase sequence. The identification point of Bgil is surrounded.

- The region of the plasmid pT4-M has a region spanning the mutation. The BglII site is ringed. The asterisks indicate deviations from the nucleotide sequence of maltase. The change in the fourth codon is a silent mutation.

c) Plasmids are not drawn to scale. Abbreviations: H = HindIII; Bc = Bell; E = EcoRI;

Bg = BglII; EV = EcoRV; Bg / Bc = BglII / Bcl junction; pEFlfiC A (dashed region), 5 'edge (promoter, + 5' leader sequence); fl ori = origin of replication of phage fl; amp = ampicillin resistance gene.

d) Plasmids are not drawn to scale. The abbreviations are the same as in Figure 7c.

Figure 8 shows the plasmid pGb-SNENS. Plasmids are shown schematically and are not to scale. Abbreviations: E = EcoRI; H = HindIII; Sf = Sfil; N = NotI; fl ori = origin of replication of phage fl; amp = ampicillin resistance gene. Arrows indicate ‘1 direction 5’ - *> - 3 ’. Oligo 3 and 4 are synthetic oligodeoxynucleotides with base sequences shown.

Figure 9 shows the construction of plasmid pGb-RB2. Abbreviations: E = EcoRI; Bg = BglII; H = HindIII; He =

hindi; B = BarnHI; Sm = Smal; P = PstI; fl ori = origin of replication of phage fl; amp = ampicillin resis -! * tens gene. The arrows indicate the direction 5 '- ^ 3'. Oligo 5 and 6 are synthetic oligodeoxynucleotides with base sequences shown. Lecture end codons TAA are underlined in all reading phases.

Figure 10 shows the construction of plasmid pGb-RBN 3. Arrows indicate the direction of transcription. Plasmids 100473 20 are schematically drawn and are not to scale. Abbreviations: Sf = Sfil; Sm = Smal; H = HindIII; fl ori = phage fl replication origin; Amp = ampicillin resistance gene; G418, Tn5 gene (under the control of the ADHI promoter) conferring resistance to G418; EV =

EcoRV; He = Hindi.

Figure 11 shows the construction of plasmid pGb-RBRRO1. Plasmid pGB-iRRO1 is fully depicted in Figure 7 and contains the maltase gene under the control of the EF1 A promoter (pEF1A) and the maltose permease gene under the control of the alcohol dehydrogenase I promoter (pADHI) (both promoters are indicated by dashed lines). Plasmid pGb-RB2 is described in Figure 9. Plasmid pGb-RBRRO1 has a fragment containing SIT4 divided into two parts ("SIT4 edges"). Plasmids are shown schematically and are not to scale. The abbreviations are the same as in Figure 10. The arrows indicate the direction of transcription.

Figure 12 shows single-step gene disruption. In step 1, plasmid pGb-RBN3 is cleaved with Sfilr.

This releases a DNA fragment that has homology to the SIT4 gene region on either side. This directs integration into the SIT4 gene (see also R.J. Roth-Stein (1983), Methods in Enzymology, 101, 202). The SIT4 gene region is marked with a dash. Both *; chromosomal alleles are shown schematically.

In step 2, the resulting strain ApGb-RBN3 is transformed with both pUT332 (uncleaved) and Sfils-truncated pGb-RBRRO1. The principle of co-transformation is well described in the literature (see AH 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 choice, the present inventors have used phleomycin resistance (plasmid pUT 332), but equally useful episomal plasmids derived from μu that confer resistance to other antibiotics such as hygromycin 21 100473 B. Plasmid pUT332 and phleomycin are commercially available from Cayla (Avenue Larrien, Center Commercial de Gros, 31094 Toulouse Cedex, France). The phleomycin resistance gene of plasmid pUT332 is derived from the transposon Tn5 and is located under the control of the yeast promoter. In step 3, the episomal plasmid pUT332 is removed from the transformants by growth in a non-selective nutrient medium (purification from the plasmid). Abbreviations: G418r = G418 resistance gene under the control of the ADHI promoter; Sf = end of the DNA fragment obtained by Sfil cleavage; MAL = modified maltase and maltose permease gene. See also Figures 9 and 11 for details; + pUT332 is the episomal plasmid pUT332.

Figure 13 shows the increase in specific activities of maltose permease and maltase as glucose leaves nutrient medium A. The curves are typical of commercial baker's yeast strains, such as strain A.

Figure 14 shows the specific activities of maltose permease in strain A and its rDNA derivatives when dough rise is simulated in nutrient medium A.

Figure 15 shows the specific activities of maltase when the dough rise is simulated using strain A and its rDNA derivatives in nutrient medium A, which contains maltose as the main source of carbon and energy; appended.

Figure 16 shows the fermentation of maltose when dough rise is simulated using strain A and its rDNA derivatives in nutrient medium A, which contains maltose as the main source of carbon and energy.

Figure 17 shows the fermentation of maltose when the dough rise is simulated using strain A and its rDNA derivatives in nutrient medium B, which contains glucose as the main source of carbon and energy.

The invention is illustrated by the following practical test results. It will be appreciated that other yeast strains and vectors may be used by one skilled in the art familiar with these methods for the purposes of the present invention. These modifications are within the scope of the present invention.

cloning techniques

For general cloning methods, reference is made to the Maniatis et al. Manual (T. Maniatis, E. F. Fritsch, J. Sambrook (1982) Molecular Cloning, A Laboratory Manual). Restriction enzymes are used according to the manufacturer's instructions and are available from either New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL), or Boehringer Mannheim (Boehringer). Generally, 1 to 5 units of enzyme are required to cleave 1 pg of DNA.

Transformation of E. coli was performed using the CaCl 2 technique (T. Maniatis et al., Supra).

Construction of Recombinant Plasmids 1) pGb-eMAL6g This plasmid is capable of self-replication in yeast and contains genes encoding maltose permease and maltase. Its construction is shown schematically in Figure 1.

Plasmid peG418 is derived from plasmid pEMBLYe23 (Baldari and G. Cesarini (1985), Gene 35, 27) and contains a fragment with the Tn5 gene between the SalI and HindIII sites (Reiss et al., EMBO J. (1984H, 3317), which confers resistance to G418 under the control of the yeast alcohol dehydrogenase I (ADHI) promoter, which is * * similar to that described by Bennetzen and Hall (JC Bennetzen and BD Hall (1982), J. Biol. Chem., 257, 3018. Plasmid peG418 was digested with HindIII, dephosphorylated with CIP, and ligated into the cleavage product obtained by digestion of pY6 with HindII and PvuI.Plasmid pY6 is described in RB Needleman 100473 23 and C. Michels. (1983) Mol. Cell Biol 2/796; RB Needleman, DB Kaback, RA Dubin, SL Perkins, NG Rosenberg, KA Sutherland, DB Forrest and C. Michels (1984) Proc. Natl. Acad. Sci USA 81_, 2811 and contains a 7.0 kb HindIII fragment with the MAL6g locus to give plasmid pGb-eMAL6g.

2) pGb-eMAL61 This episomal plasmid derived from 2 contains a gene encoding maltose permease. The construction of this plasmid is shown schematically in Figure 2.

Plasmid pGb-eMAL6g contains two BglII sites, each in the malt gene. This 1.4 kb BglII fragment was removed from plasmid pGb-eMAL6g by BglII digestion and subsequent re-insertion under dilute conditions to promote intramolecular ligation. Such removal 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 2yu-derived episomal plasmid contains DNA that encompasses MALβ function (regulatory protein gene or MAL regulator). The construction of this plasmid is shown schematically in Figure 3.

A KpnI-SalI fragment containing the regulatory protein gene was isolated from plasmid p21-40 (R.B. Needleman and C. Michels (1983), supra). This fragment was homogenized using T4 DNA polymerase and Klenow DNA polymerase and then cloned into the filled HindIII site of plasmid peG418.

4) pGb-M6g (4-9) This plasmid is a promoter deletion mutant made by the 100473 24 deletion between the maltose permease gene and the maltase gene when the genes are transcribed in opposite directions (see Figure 4). This region contains the promoters of both genes (S.H. Hong and J. Marmur (1986) Gene 41, 75). This deletion mutant has been prepared to replace the original promoters. The construction consisted of the following steps: a) An approximately 7.0 kb HindIII fragment containing the maltase and maltose permease genes (see also Figure 1) was cloned into the HindIII site of plasmid pTZ19R. This plasmid is commercially available (Pharmacia). The result was pGb-M6g.

b) Plasmid pGb-M6g was linearized with Stulr, which cleaves from the intergenic region. The ends generated by Stuls served as starting points for the Bal31 exonuclease, which gnawed out the region between the genes so that the promoters (or parts of them) were deleted. The Stul site is closer to the maltose permease gene (S.H. Hong and J. Marmur (1986), supra). Bal31 incubation was performed as described by Maniatis et al. (T. Maniatis, E. F. Fritsch, and J. Sambrook (1982), supra). At appropriate times, a sample was removed from the reaction and the resulting samples were incubated with Klenow DNA polymerase to obtain flat ends. Synthetic linkers with multiple restriction sites were then attached to the ends.

The following complementary oligodeoxy ribonucleotides were used: 1. 5 'GATATC CTCGAG AGGCCT A 3' 2. 3 'CTATAG GAGCTC TCCGGA TGCGC 5' • »

In the double-stranded form, cleavage sites are generated for EcoRV (GATATC), Xholr (CTCGAG), Stul: AG (AGGCCT), and the MluI site (ACGCGT) is formed at the junction of the stepped ends. Following the kinase reaction, the linkers were ligated into Bal31-treated DNA according to the manual of T. Maniatis 100473 et al (supra). The reaction mixture was then incubated with Mlulr and chromatographed through a 5 ml Sepharose Cl-2B column to separate the uncoupled oligodeoxynucleotides from the DNA fragment. Fractions containing this linear DNA were pooled, pooled into plasmid DNA and transferred to a bacterium.

c) The set of deletion mutants thus obtained was subjected to sequence analysis. To this end, double-stranded plasmids were converted to single-stranded DNA by superinfection with helper phage (according to the manufacturer's instructions). Single-stranded templates were extracted by standard M13 methods for use in dideoxy sequencing (F. Sanger, S. Nicklen, and A.R. Coulson (1977) Proc. Natl.

Acad. Sci. 2i / 5463). A synthetic oligodeoxynucleotide (5'-GAATTCGGTAGCGTTCACGC-3 ') complementary to the DNA sequence close to the ATG start codon of the maltose permease gene was used as a primer. Its orientation is such that the sequence is read toward the promoter (see also Figure 4).

The deletion mutant from which most of the maltose permease promoter had been deleted was selected for further experiments. The maltase promoter (or part thereof) is still present (Note that the Stul site, which serves as the starting point for exonuclease treatment, is located asymmetrically in the intergenic region). Figure 5 shows the sequence of the deletion site of the mutant pGb-M6g (E-9).

This was compared to the newly determined sequence of this entire region (S.H. Hong and J. Marmur (1986), supra). One difference in the wild-type sequence has been observed. relative to that sequence: at position -878 (numbering S.H.

According to Hong and J. Marble (1986), supra, C is not present in our sequence. Thus, DNA cannot be cleaved by HpaI or Hindi at this point.

Plasmid pGb-M6g (A-9) is the starting plasmid when other 100473 26 promoters are fused to both the maltose permease gene and the maltase gene (see below).

5. pGb-iA32 / G418 This plasmid is an integrative yeast plasmid. It contains the maltose permease gene linked to the alcoholide hydrogenase I promoter and part of its 5 'leader sequence. The construction of this plasmid is as follows (Figure 6).

a) Plasmid pTZ19R / ADHI contains a 1.4 kb BamHI fragment with an ADHI promoter starting at position -15 relative to the AUG codon (J.L. Bennetzen and B.D. Hall (1982) J. Biol. Chem. 257, 3018). A 700 bp EcoRV-HincII fragment was isolated from this plasmid and ligated into EcoRV-digested pGb-M6g (9). The plasmids thus obtained were analyzed by restriction enzyme cleavage and the orientation of the promoter was confirmed by dideoxy sequencing using single-stranded templates (see

Figure 6). The same oligo primer as described in 4c was used.

As a result of the cloning method of the ADHI promoter fragment, the plasmid pTZ19R multilinker (BamHI-Hindi)

CU

part is present between the maltose permease gene and the AHDI promoter. The structure and sequence of plasmid pGb-A32 are shown in Figure 6a.

b) Plasmid pGb-A32 was designated the dominant marker G418res. The 1.9 kb EcoRV / HincII fragment contains the Tn5 gene, which confers G418 resistance under the control of the alcohol dehydrogenase I promoter.

; This fragment was isolated by plasmid 153-215 AK EcoRV / HincII digestion. The EcoRV / HincII fragment was cloned into the SmaI site of plasmid pGb-A32.

Thus, plasmid pGb-iA32 / G418 was obtained.

27 100473 6) pGb-iRRol This plasmid is an integrative plasmid. It contains the maltose permease gene under the control of the alcohol dehydrogenase I promoter and the maltase gene extension factor EF1 under the control of the promoter. The cloning method is shown in Figure 7. The method was as follows: The maltase gene contains a Bell site around the fifth amino acid codon. Therefore, the region encoding EFlofA was mutated so that the first five amino acids became identical to those of the maltase protein. At the same time, a BglII site was placed in place of the BclI site. Conversion of the BclI site to the BglII site results in a silent mutation at the fourth codon. Through BglII / BclI ligation, the EF10fA promoter and leader sequence could be ligated to the rest of the malt gene. The method consisted of the following steps: a) The starting plasmid was pYEF46 (S. Nagata, K. Nagashi-ma, Y. Tsunetsugu-Yokota, K. Fuyimura, M. Miyaraki and Y. Kaziro (1984) EMBO J. 3, 1825), which contains the entire gene encoding EF10 (A. A 2.5 kb BglII fragment spanning this gene was isolated and cloned into the BamHI site of plasmid pTZ19R. Clone pT4 (see Figure 7a) was picked and used for oligodeoxynucleotide-directed mutagenesis.

• b) After superinfection with helper phage, single-stranded (ss) DNA of pT4 was isolated. 400 ng of ssDNA, 400 ng of heat-denatured pTZ19RxBamHI, and 100 ng of mutation oligodeoxynucleotide (see Figure 7b) were incubated for 10 minutes at 56 ° C in a volume of 10 with 7 mM Tris HCl pH 7.5; 50 mM NaCl and 7 mM MgCl 2 · After incubation for 10 minutes at room temperature, the synthesis and ligation of the second strand was started by adding 1 μl of Klenow DNA polymerase (2 units), 1 μl of T4 DNA ligase (4 units), 1 μl of TMD buffer (200 mM Tris HCl pH 7.5, 100 mM MgCl 2, 100 mM DTT),; 4 μl of 2.5 mM dNTP mixture, 1 μl of 10 mM ATP and 2 μl of water.

28 1 0 0 4 7 3

The final volume was 20 μl, which was incubated for 16 hours at 17 ° C, after which the mixture was transformed into E. coli JM101. Mutants were screened by colony hybridization using a kinased oligodeoxynucleotide specific for the mutant (see Figure 7b). This screening oligomer 5'-GACTATTTCAGATCTTC-3 'was complementary to the imported maltase codons and edge nucleotides. Hybridization was performed for 16 hours in 6xNET buffer (1xNET = 0.15 M NaCl, 0.015 M Tris HCl pH

7.5, 0.001 M EDTA) at 25 ° C. Post-hybridization washes were performed in the same mixture at the same temperature (3 x 10 minutes) and then washed in 3xNET buffer at 25 ° C. Several positive colonies were further analyzed. BglII cleavage confirmed the presence of the BglII site. In addition, single-stranded DNA was isolated from a mutant containing the BglII site (pT4-M) and subjected to dideoxy sequence analysis using a synthetic 17-mer primer complementary to nucleotides 87-103 of the EF1 * A gene (p. Nagata, K. Nagashima, Y. Tsunetsugu-Yokota, K. Fuyimura, M. Miyaraki, and Y. Kaziro (1984) EMBO J., 3, 1825) (5'-CAATACCACCACACTTG-3 '). The sequence result obtained confirmed that the desired mutations had been obtained.

c) The next step was to isolate from the plasmid pT4-M the EF1 * A promoter / maltase NH2-terminal segment "and insert it by joining the BglII / BclI-terminated ends to the rest of the maltase gene. This restores the maltase coding region of the EF1 ° tA. downstream of the promoter and leader sequence (see Figure 7c) Plasmid pGb-M6g (.Ä-9) was used to transform GM113, an EL_ coli Dam strain, to use the BclI- * site (which is sensitive to methylation) (GM113: thr ~ .leuB6, proA2. tris-4. metB1, IacY1, galK2, ara-14. tsx33. thi-1, thyA12, deoB16, supE44, rpsL260, Dam ~ 3). / HindIII fragment containing the maltase gene except for the part immediately at the N-terminus Plasmid pT4-M was digested with BglII / HindIII and a large 4.1 kb fragment was isolated (Tl HI i I Jt 100473 29) ( EF1 * A promoter + N1 terminus of the maltase gene) .The two fragments were ligated together to give pGb-EFMT-3. Sequence analysis confirmed that all mutations introduced were correct.

d) Finally, pGb-EFMT-3 was digested with EcoRV and HindIII to purify the 4.8 kb fragment with the EF1 A / maltase promoter / gene. An approximately 8.3 kb HindIII (partial) / EcoRV fragment was isolated from plasmid pGb-iA32 / G418 (see Figure 6). This includes the pTZ19R backbone, the ADHI / maltose permease promoter / granule gene segment, and the ADHI / G418 segment. Both sequences were joined together to give pGb-iRRol (see Figure 7d).

7) pGb-RB2 This plasmid serves as a cloning tool to integrate DNA sequences into the SIT4 gene of Saccharomyces cerevisiae. Construction of this plasmid consists of the following steps (see Figures 8 and 9).

a. Plasmid pTZ19R was digested with EcoRI and HindIII to replace its multilinker with a 40 nucleotide sequence of synthetically prepared DNA. This DNA sequence was prepared by base pairing with synthetic oligonucleotides 3 and 4 (see Figure 8). This short fragment contains a staggered EcoRI end and a HindIII end. Cloning of this DNA fragment into plasmid pTZ19R does not restore the EcoRI site or the HindIII site. The synthetic DNA fragment contains, at the boundaries, restriction sites for NotI and SfiI and an EcoRI site in the middle, as indicated in the figure. The plasmid thus obtained was named pGb-SNENS.

b. An EcoRI fragment containing the SIT4 gene (Gottlin-Ningaja Kaback, supra) was isolated from plasmid pLF24 (also known as pLN420) and cloned into the EcoRI site of plasmid pGb-SNENS to give pGb-Spons: 31. Since there are no comparable restriction sites 100473 30, its direction is unknown.

c. Plasmid pGb-Spons 31 contains the only BglII site within the SIT4 gene. This can be used as a site where any DNA sequence that is desired to be inserted into the SIT4 gene by gene replacement can be cloned. To facilitate modification in cloning, the SI-T4 gene was provided with a synthetic DNA sequence containing several single restriction sites. The construction of plasmid pGb-RB2 is shown schematically in Figure 9.

The synthetic DNA fragment has two stepped BglII ends. Its orientation in plasmid pGb-RB2, as indicated in Figure 9, is based on restriction enzyme analysis of plasmid pGb-RBN3 (see next section).

8) The 1.9 kb EcoRV / HincII fragment of pGb-RBN3 containing the res G418 gene under the control of the ADHI promoter (see also construction of plasmid pGb-iA32 / G418) was cloned into the SmaI site of plasmid pGb-RB2. Thus, plasmid pGb-RBN3 was obtained (Figure 10).

9) pGb-RBRRO1 ·; 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 EF1 A promoter. Both are located in an 8.3 kb fragment cloned into the SIT4 gene. The construction of this plasmid is shown schematically in Figure 11. To this end, pGb-iRRO1 was digested with HindIII and ligated with HindIII. plasmid pGb-RB2 (treated with calf visceral phosphatase). Thus, plasmid pGb-RBRRO1 was obtained.

10) pGb-RBREGO1 This plasmid contains an alcohol 100473 under the control of the alcohol dehydrogenase promoter for the MAL regulatory gene.

I. Plasmid pGb-RBREGO1 can be constructed as follows. A SalI fragment containing the regulatory protein gene is isolated from plasmid p21-40 (R.B. Needleman and C. Michels (1983), supra). This fragment is cloned into the SalI site of plasmid pTZ18R. This plasmid is commercially available (Pharmacia). Its orientation is such that the promoter region of the MAL regulatory gene is close to the T7 promoter sequence of pTZ18R. Using the pTZ18R sequence primer and other freshly prepared primers based on the resulting DNA sequence, the promoter region of the MAL regulatory gene and a portion of the coding region for the N1 end of the gene can be sequenced. This locates the AUG start codon. If there are no useful restriction sites in the promoter region near the AUG start codon, an appropriate site (e.g., BglII) can be introduced into this region using oligonucleotide and conventional methods for site mutation (see also construction of recombinant plasmids, section 5b).

Following such mutation, the BglII-SalI fragment (containing the MAL regulatory gene without a promoter) and the EcoRV-BanHI fragment (containing the ADHI promoter, see also Figure 7d, pGb-iRRO1) can be co-inserted into plasmid pGb-RB2 (see Figure 9). ) so that the MAL regulatory gene becomes under the control of the ADHI promoter. This gives plasmid pGb-iRBREGO1. By performing sequence analysis by the dideoxy strand method using oligonucleotides and an ssDNA template, the correct completion of the cloning steps and the direction of the promoter can be easily ascertained.

It will be appreciated that the plasmid constructs described above are merely examples to illustrate the present invention. Other promoters (preferably derived from Saccharomyces (or other portions of the promoter used), other integration loci, and other combinations of maltase, maltose permease, and MAL regulator 32 100473 may be used (either using one promoter of its own or another promoter, preferably a Saccharomyces promoter). or more integration sites, resulting in an optimal ratio of maltase to maltose permease activities throughout the anaerobic fermentation.

Yeast transformation

Transformation of yeast strains was performed by the method of Ito et al. (H. Ito, Y. Fukuda, K. Murata, A. Kimura (1983), J. Bacteriology 153, 163-168). In the method, Saccharomyces yeast is grown in standard yeast nutrient medium to a density of 1-25, preferably in the range of 4-10 ODg10. The yeast cells are then harvested, washed and pretreated with chaotropic ions, in particular alkali metal ions lithium, cesium or rubidium, in particular as chloride or sulphate, and in particular lithium salts with a concentration of about 2 mM to 1.0 M, preferably about 0.1 M After incubation of the cells for about 5 to 120 minutes, preferably about 60 minutes, with chaotropic ion (s), the cells are incubated for a short time at a moderate temperature, usually about 20 to 35 ° C for about 5 to 60 minutes. Preferably, polyethylene glycol is added to a concentration of about 25-50% so that the entire mixture is diluted by adding the same volume of polyethylene concentrate to result in the desired final concentration. The polyethylene glycol has a molecular weight of about 2,000 to 8,000 daltons, preferably about 4,000 to 7,000 daltons. The incubation time is usually relatively short, about 5-60 minutes. Preferably, the incubation mixture is heat treated for about 1-10 minutes at about 35-45 ° C, preferably at about 42 ° C. Any marker can be used to select transformants, such as phleomycin (D. Genilloud, M. C. Garrido, F. Moreno (1984) Gene 32, 225), hygromycin. B (Gritz et al. (1983) Gene 25, 1978) and aminoglycosyl-

I: I dill I I I 11 100473 33 dia G418 (Jiminez et al. (1980), Nature, 287, 869).

When yeast cells were transformed with the integrating plasmid, integration was directed to the MAL locus using BglII-digested DNA. The integrating plasmids used contained two BglII sites, both located in the malt gene 1.4 kb apart. This creates double-stranded breaks that are recombinant and stimulate interaction with homologous chromosomal DNA. The gap is repaired by chromosomal information during the integration event (T.L. Orr-Weaver, J.W. Szostak, R.Rothstein (1981) Proc. Natl. Acad. Sci. USA 7j3, 6354). The integration event yields one or more copies of the plasmid vector (J.W. Szostak, R. Cou (1979) Plasmid 2, 536). The exact copy number contained in these transformants used in the carbon dioxide production experiments has not been determined.

A way was developed to obtain stable yeast transformants that do not contain heterologous DNA. Examples of heterologous DNA are pTZ19R vector DNA and selection genes that confer resistance to the antibiotics G418, phleomycin or hygromycin B. Briefly, the experimental setup was as follows (Figure 12): 1) A single-step disruption of the SIT4 gene was performed here by transforming the yeast with plasmid pGb-RBN3 digested with SfiI. Transformants were selected based on G418 resistance. Thus, strain ApGb-RBN3 was obtained in which the SIT4 gene ar was replaced by the SIT4 gene centered by the G418r gene, which is a resistance gene under the control of the ADHI promoter.

• 2) Strain ApGb-RBN3 was used as a host strain in co-transformation, where transformation was performed with plasmid pUT332 and plasmid pGb-RBRRO1 digested with Sfil. Plasmid pUT332 is an episomal plasmid derived from 2j and contains a gene conferring phleomycin resistance. In this co-transformation, the first selection was made in plates containing phleomycin (30 pg / ml). In a certain percentage of these phleomycin-resistant yeast cells (on the order of 0.1 to 1%), the disrupted SIT4 gene (containing pADHI / G418r) has been replaced by the co-transformation fragment SIT4, which contains the modified maltose permease and maltase genes. This second gene replacement event resulted in a yeast that was again sensitive to G418. To select yeast cells in which this second gene replacement had occurred, phleomycin-resistant transformants were plated in plates containing G418 (300 ug / ml). In cells that did not grow, the SIT4 gene inter-sequence G418r gene had been replaced with modified MAL genes inter-SIT4 gene sequences.

3) Phleomycin-resistant, G418-sensitive transformants were then purified from the episomal plasmid pUT332 by growth in a non-selective medium (i.e., without phleomycin) for 10 to 20 generations. The transformant thus obtained is sensitive to both phleomycin and G418 and does not contain prokaryotic sequences. All integration events were confirmed at the DNA level by Southern blotting (data not shown).

The strain thus obtained could be used as a host in subsequent transformations using a different integration locus and / or - if the yeast strain is diploid or polyploid - 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 targeting sites for gene replacement. This eventually yielded the transformant ApGb-p2RBRR01 # 1. Both alleles. use in integration most likely also increased the genetic stability of the transformant, as another integration event removed the polymorphism from this locus. Such poly-

»I 1 li i i Hl i i i -H

100473 35 morphological regions are sensitive to gene conversions that may result in loss of integrated sequences. The transformant thus obtained was analyzed by Southern blotting and gave the hybridization pattern expected as a result of disruption in both SIT4 genes.

The constructed vector pGb-RB2, which served as the starting plasmid for the construction of plasmids pGb-RBN3 and pGb-RBRRO1, has several useful properties when viewed from the following considerations: 1. Often, the DNA sequence must be integrated by integrating the yeast promoter with another ( yeast) to the coding region of the gene. Of course, gene substitutions are easily made to occur only if the transforming fragments have sequences on each side that are homologous to the genomic alignment sequence. Thus, the DNA sequence must be inserted at the 3 'end of a coding region (see above) derived from the same gene as the selected promoter. The disadvantage of this approach is that it requires many cloning procedures because the same promoter is not always used. In addition, a strong promoter is often selected that is derived from a gene that functions at desired stages (during vegetative growth, during anaerobic fermentation, etc.). Once a genetically engineered fragment is integrated, one copy of the gene has lost its ability to function.

Thus, the present inventors have sought to direct the targeting of gene replacement to a locus that does not occur during vegetative growth. The present inventors have selected the SIT4 gene (sporulation-induced transcribed sequence), the expression of which has been extensively studied by Gottlin-Ninga and Kaback (supra), but of course other SIT genes and non-coding segments in general are also suitable. When the desired DNA construct is cloned into the SIT4 gene, 100473 36 of the two halves of the SIT4 gene thus generated act as homologous ends in recombination.

It can be argued that the chromosomal region in which the gene is located is transcriptionally inactive during vegetative growth due to a silencer analogous to that described for the HMR locus (A.H. Brand et al. (1985) Cell 41, 41-48). This silencing DNA represses promoter-regulated transcription regardless of mating-type promoters and can affect promoters up to 2600 bp away. However, Gottlin-Ninga and Kaback (supra) have shown that the HIS3 gene is able to function during vegetative growth when integrated into the SIT4 gene.

2. To facilitate cloning procedures, the SIT4 gene is provided with a synthetic DNA sequence containing several single restriction sites (a multi-linker with cloning sites). The multi-switch also contains stop codons on the heat side, which enable reading in all possible reading phases (see Figure 9). This is a backup valve that stops reading any possible recombinant transcript that may be synthesized across the junction. Such a recombinant transcript may otherwise encode a protein whose effect is unknown.

3. In order for homologous integration to occur, the DNA segment to be integrated contains restriction sites for NotI and Sfilr at each end, each of which recognizes an 8 bp sequence. The frequency of occurrence of these cleavage sites is very low and thus it is highly unlikely that the DNA segment to be cloned into the multi-linker contains both cleavage sites. Thus, when a DNA segment is cloned into the SIT4 gene of plasmid pGb-RB2, cleavage with NotI or Sfil releases a DNA fragment that can recombine by interacting with the inherited homologous sequences 100473 37, i. SIT4 locus. Although the very ends are part of the NotI or Sfil site and are therefore not homologous, other studies have shown - apparently due to limited exonuclease cleavage in the cell - that these sequences are deleted (e.g.

H. Rudolph, J. Koenig-Ranseo, and A. Hinnen (1985) Gene 36, 87-95).

4. The starting plasmid used is the commercially available plasmid pTZ19r, which is a derivative of pUC19. This vector has the advantages of having a high copy number and having an origin of replication of the single-stranded phage fl. For this reason, it is very easy to isolate - after infection with helper phage - single-stranded DNA and to confirm DNA sequences extending across junctions using primers. This greatly facilitates the detailed characterization of the engineered DNA.

It is clear that these improvements can be applied to other yeasts in addition to baker's yeast.

Measurements of carbon dioxide production (a) In synthetic dough medium (test A)

Yeast cells were incubated in YEPMS medium (1% yeast extract; 2% bactopeptone; 3.75% maltose; 1.25% sucrose plus 200 μg / ml G418). Grown at 30 ° C until the late logarithmic step. Yeast cells were harvested from 6 ml of culture and resuspended in 8.8 ml of synthetic dough medium. The composition of this medium was as follows (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; (NH4) 2SO4 0.67 g; casino acids 2.07 g; citric acid 4.03 g; Na 2 citrate 44.25 g; vitamin B1 9.2 mg; vitamin B6 9.2 mg; nicotinic acid 46 mg; Ca - (+) - pantothenate 18.2 mg; biotin 0.23 μg. The suspension was allowed to equilibrate for 10 minutes at 28 ° C in a water bath while stirring at 100473 38. This burette was filled with a solution containing 20 ml per liter of indicator solution (1 g of methyl flag; 0.5 g of methylene blue dissolved in 1 liter of 96% ethanol), 40 ml of 1 N sulfuric acid and a little copper sulphate (CuSO 4) (dissolved in nitric acid). ).

The displacement of the volume of this solution in the burette is a measure of carbon dioxide production measured within 165 minutes.

In each test set-up, the carbon dioxide production values have been corrected from the prevailing pressure and temperature to standard conditions of 28 ° C and 760 mm Hg, respectively. In addition, a correction was made for the amount of yeast. Colorimetric readings of the cultures at 600 nm have been used as a correction factor to make the yeast amount of each carbon dioxide measurement consistent with each other.

b) In the dough (tests B and B ')

For compressed yeast grown with molasses: carbon dioxide yield curves were determined in a dough without added sugar (lean dough) (test B) and in a dough with 30% sugar (test: B ') · The lean dough was prepared as follows: 1 g of compressed yeast (containing 28.5% dry matter), 34 ml of brine A (1.25 g of sodium chloride dissolved in 34 ml of distilled water) and 62.5 g of flour were mixed in a Hobart apparatus for 30 seconds at speed 1 and. 2 minutes at speed 2 so that a well-developed. now the dough.

The 30% sugar dough contained 2.0 g of compressed yeast (28.5% dry matter), 34 ml of brine B (0.938 g of sodium chloride in 34 ml of distilled water), 62.5 g of flour and 18.75 g of sucrose ( i.e. 30% sugar based on flour). Mixing was performed on 100473 39 as well as lean dough mixing.

The dough was then transferred to a round bottom flask. The gas production measurement was started 7.5 minutes after mixing by connecting the flask containing the dough to a gas burette (see a) and the measurement was performed in 165 minutes at 28 ° C. If the dry matter content of the compressed yeast differed from the above, the measured carbon dioxide production capacity was corrected by multiplying the carbon dioxide production capacity by the ratio of the required dry matter content to the actual measured dry matter content. In each experimental set-up, the CO2 production values obtained have been corrected for ambient temperature and pressure to correspond to 28 ° C and 760 mm Hg. In some cases, additional calculations have been performed to correct the gas production values obtained according to the nitrogen content of the yeast batch (nitrogen percentage indicates protein content). Similar percentages were observed, although no latter correction was made.

(c) in the dough (tests C and C),

The carbon dioxide production capacity of dry yeast, such as instant dry yeast prepared according to U.S. Patent Nos. 3,834,800 and 4,348,877, was determined in a dough to which no sugar had been added (Test C) or in a dough to which 30% sugar had been added (Test C). These experiments were performed in the same way as experiments B and B 'except that experiments C and C used 300 mg of dry yeast (containing 96% dry matter) and 600 mg of dry yeast (containing 96% dry matter), respectively. Prior to the experiment, the yeasts were mixed with flour and incubated for 10 minutes at 28 ° C.

Enzymatic analysis

The ability of yeast cells to transfer maltose was determined using (U-C) maltose at a concentration of 15 mM as a substrate at 30 ° C. Details are published by R. Ser- 100473 40 rano (supra). Maltase (E.C. 3.2.1.20) was determined using p-nitrophenyl-o (-D-Glu-copyranoside in cell-free extracts as substrate) according to H. Halvorson and E.L. Elias, Biochim. Biophys. Acta (1958) 22-28.

Substrate consumption and product formation in a liquid nutrient medium

The loss of maltose and glucose from liquid nutrient media was quantified using standard HPLC methods. One liter of nutrient medium contained 100 g of maltose, 10 g of glucose, 3.0 g of NH 4 O 2, 4.0 g of MgSO 4, 4 g of Ki 2 PO 4, 4 g of amino acid and (Difco), 4 g citric acid H90, 45 g trisodium citrate.2Ho0, 10 mg vitamin B1, 10 mg vitamin B6, 40 mg nicotinic acid, 20 mg Ca-D (+) - pantothenate and 0.02 mg biotin The pH was adjusted to 5, 7. 2 ml of nutrient medium was added to the yeast suspension (20 mg of yeast by dry weight / 2.0 ml of distilled water) The resulting mixture, designated mixture A, was incubated at 28. Mixture B was like mixture A, but contained 20 times more glucose.The experiment was performed under anaerobic conditions.

A protein assay

Protein from cell-free extracts and whole cells was determined by the microbial method (J. Goa,

Scand. J. Chim. Lab. Invest. (1953) 5, 218). Ovalbumin served as standard.

: Shelf life

Compressed yeast was stored in sealed plastic containers at 23 ° C for 4 days. Shelf life is expressed as a percentage of the gas production capacity remaining after this period.

100473 41

Manufacture of compressed yeast

The yeast strain was grown in a series of fermentors. Cells were cultured in 10-liter laboratory fermenters with a net volume of 6 liters. During the fermentation, the pH and temperature were maintained at the desired values with the aid of automatic adjustment. The fermentation was based on the methods described in G. Butschek and R. Kautzmann, Die Hefen,

Band II Technologia der Hefen, ss. 501 - 591 (1962) Verlag Hans Carl, Niirnberg, West Germany and G. Reed and H.J.

Peppier in Yeast Technology, the AVI Publishing Comapny Inc., Westport, Connecticut, USA (1973). In particular, the culture conditions for the final fermentation were as follows: the molasses used consisted of 80% by mass of sugar beet molasses and 20% by mass of sugar cane molasses, calculated on the basis of 50% sugar; the required amount of phosphate was added in the form of monoammonium phosphate before inoculation; during fermentation, the temperature rose from 28 ° C to 30 ° C according to Table 1; during the fermentation, nitrogen was introduced as a 10% aqueous solution of ammonia according to Table 1; The pH was maintained at 5.0 for the first 8 hours of fermentation and then raised to 6.2 according to Table 1 at the end of the fermentation; Per 1 kg of molasses containing 50% fermentable sugars, 12 mg of vitamin B1 was added before inoculation.

The yeast obtained by this fermentation was concentrated and washed with tap water in a nozzle centrifuge for laboratory use. The yeast masses were compressed to a dry matter content of 26-32%.

The protein content obtained (% N x 6.25) ranged from 42 to 55% on a dry weight basis due to the different large amounts of ammonia added during the fermentation.

42 100473

table 1

Fermentation recipe when baking yeast was produced by the batch feed method

Hours in. Add pH T Added from the amount of molasses ° C Amount of niacry (% size (% of total added amount added)) <0 75 28.0 0 0-1 - 5 28.0 0 1- 2 5 5 28 .0 0 2- 3 6 5 28.5 1 3- 4 8 5 28.5 7 4- 5 8 5 29.0 11 5- 6 8 5 30.0 11 6- 7 10 5 30.0 12 7- 8 10 5 30.0 15 8- 9 10 5.3 30.0 17 9- 10 10 5.6 30.0 17 10- 11 10 5.9 30.0 10 11-12 8 6.2 30.0 0

Example 1

Carbon dioxide production of a yeast transformed with a 2β-derived plasmid containing genes derived from the MAL6 locus

Commercially available yeast strains A and C were transformed with plasmid pGb-eMAL6g (maltose permease and maltase, see Figure 1), plasmid pGb-eMAL61 (maltose permease, see Figure 2) or plasmid pGb-eMAL63 (MAL regulator, see Figure 1). 3). The MAL genes still contain their original promoter. The naming system for transformed yeast cells is as follows:

ApeG418 refers to strain A transformed with peG418. Other transformed strains are labeled accordingly. The effect of these plasmids on carbon dioxide production in the synthetic dough medium is summarized in Table 2. The reference strain was the host strain transformed with the starting plasmid peG418 (see Figure 1), as the present inventors have found that the presence of multiple copies of the plasmid alone has a negative effect on gas.

All transformants show a significant increase in carbon dioxide production compared to the control.

When a copy of both the maltose permease gene and the maltase gene is present, the largest increase in production is achieved, about 40% for strain A and 18% for strain C.

Transformants ApGb-eMAL6g and CpGb-eMAL6g were also tested in doughs without added sugar.

In this case, the yeast was grown by feeding Melas in batches.

Again, a significant increase in carbon dioxide production was achieved.

Table 2 • 44 100473

Gas production of strain A and strain C after transformation with a plasmid derived from 2α. Carbon dioxide production was measured in synthetic dough medium (see experimental methods) and corrected to a dry weight of 285 mg. The results are the averages of several experiments.

You carry 100 minutes to 165 minutes

ApeG418 100 100

ApGb-eMAL6g 157 141

ApGb-eMAL61 144 123

ApGb-eMAL63 119 115

CpeG418 100 100

CpGb-eMAL6g 121 118

CpGb-eMAL61 117 114

Table 3

Relative gas production of strains A and C when the strains were transformed with plasmid peG-41 & and pGb-eMAL6g derived from 2, respectively. Carbon dioxide production was corrected to a dry weight of 285 mg.

Strains 60 mi- 100 mi- 120 mi- 165 mi minutes minutes minutes minutes ApeG418 100 100 100 100

ApGb-eMAL6g 125 125 125 121 *: Cpe418 100 100 100 100

CpGb-eMAL6g 151 141 138 129 li: SS: i Itll I t »t» 100473 45

Example 2

Carbon dioxide production of yeast strains transformed with an integrating plasmid containing the genetically engineered maltase gene and / or the maltose permease gene

Basic yeast strain A has been transformed with plasmid pGb-iA32 / G418 (main feature: ADHI / maltose permease;

Figure 6) or plasmid pGb-iRRol (main feature: ADHI / maltose permease and EF1 <* A / maltase; see Figure 7).

Base strain A and each integrant-containing transformant were grown by adding molasses in batches as well as in commercial aerobic fermentation (see Table 1). After the cells were harvested, their carbon dioxide production was measured by a dough standard experiment without added sugar. The gas production obtained by measurements in this experiment is summarized in Table 4. The integration of plasmid pGb-iA32 / G418 into the chromosome of strain A significantly improves gas production. The relative improvement varies slightly depending on the time of measurement (see Table 4). When, in addition to the modified maltose permease gene, the modified maltase gene is integrated into the chromosome of commercial strain A using the plasmid pGb-iRRol, gas production is further improved. In this typical experiment, the lean dough produces about 30% more carbon dioxide after 165 minutes, which corresponds to about 410 ml carbon dioxide / 285 mg yeast by dry weight.

In the dough to which 30% sugar had been added, no differences in carbon dioxide production were observed with the transformants compared to the base stock (approximately 190 ml carbon dioxide / 285 mg yeast by dry weight).

The improvement in elevation activity achieved is maintained when stored at 23 ° C. The loss of lifting capacity in storage is essentially the same for base strain A and new strains (see Table 5).

Table 4 46 100473

Relative gas production of strain A and its genetically engineered derivatives with a modified maltase and / or maltose permease gene. The gas values have been corrected to a dry weight of 285 mg. No sugar was added to the dough.

Stock 60 mi- 100 mi- 120 mi- 165 mi minutes minutes minutes minutes A 100 100 100 100

ApGb-iA32 / G418 113 115 115 111

ApGb-iRRol 131 136 138 133

Table 5

Stability of strain A and its genetically engineered derivatives with a modified maltase and / or maltose permease gene. The lifting capacity was measured in the same way as in Table 4 after the compressed yeast was first stored for 4 days at 23 ° C.

Stock shelf life (% of original lifting capacity) A 90

ApGb-iA3 2 / G418 91

ApGb-iRRol 88

Example 3

Carbon dioxide production of a yeast strain containing genetically engineered maltase gene and maltose permease gene but no heterologous DNA

Base strain A was genetically engineered by placing the 100473 47 pADHI / maltose permease gene and the pEF1 / A / maltase gene on the SIT4 gene on each homologous chromosome. This strain, named ApGb-p2RBRR01 * 1, was constructed using the methods and plasmids described above (see sections describing the transformation and construction of plasmids pGb-RBN3 (Figure 10) t / and pGb-RBRRO1 (Figure 11) and the general trans -formation scheme in which gene replacement is performed). Base strain A and its homologous transformant ApGb-p2RBRR0141 were grown by batching molasses as well as in commercial aerobic fermentation (see Table 1). After the cells were harvested, carbon dioxide production was measured by a standard dough experiment without the addition of sugar.

Table 6 summarizes the results obtained. In a "lean" dough, the improvement is about 18% after 165 minutes, which corresponds to a level of about 367 ml carbon dioxide / 285 mg yeast by dry weight. This strain contains, in duplicate, both the maltose permease gene under the control of the ADHI promoter and the maltase gene under the control of the EF1A promoter. Table 4 shows that the improvement in the yield of the strain ApGb-iRRO1 is about 30%. An initial estimate of the number of pGB-iRRO1 molecules integrated into the MAL locus of strain A results in at least 3 integrated plasmid molecules. When the copy number of the modified maltose permease gene and the maltase gene is further increased in the strain ApGb-p2RBRR01 (for example by integration with other sporulation-specific genes), a homologous transformed yeast strain with at least as good gas production capacity can be obtained. than the strain ApGb-iRRO1.

Table 6 48 100473

Relative gas production of strain A and a genetically engineered homologous derivative with a modified maltase gene and a maltose-sipermase gene. The gas values have been corrected to a dry weight of 285 mg.

Stock 60 mi- 100 mi- 120 mi- 165 mi minutes minutes minutes minutes A 100 100 100 100

ApGb-p2RBRR011l 124 128 127 118

Example 4

Enzymatic activities and substrate utilization capabilities of yeast strains transformed with an integrating plasmid containing the genetically engineered maltase gene and / or maltose permease gene

As described above, the expression of maltose permease and maltase is susceptible to the inducing effect of maltose and glucose repression.

This phenomenon can be seen in Figure 13, which relates to wild-type cells representing strain A. The specific activities of maltose permease: and maltase do not increase until most of the glucose has been used.

The activity of maltose permease at the beginning of dough rise increases when a modified maltose permease gene (strain ApGb-iA32 / G418) is introduced into the yeast genome, as shown in Figure 14.

. Surprisingly, maltase activities also increased in that structure (Figure 15). This new strain, ApGb-iA32 / G418, fermentes maltose faster than base strain A in nutrient medium A, which contains maltose as the main source of carbon and energy (Fig. 16). In nutrient medium B, which contains glucose as the main source of carbon and energy, this effect was weaker (Figure 17).

When the modified maltose permease gene and the modified maltase gene were integrated into the chromosome, the strain ApGb-iRRol was obtained. This strain fermented maltose at an even higher rate in nutrient medium A (Fig. 16) and also in nutrient medium B (Fig. 17). Despite high extracellular glucose levels, this new strain metabolizes significant amounts of maltose. Strain ApGb-iRRol showed higher specific activities of maltase and maltose permease than base strain A and strain ApGb-iA32 / G418 (Figures 14 and 15) during dough raising. 1

Claims (33)

A transformed yeast belonging to the genus Saccharomvces and which has, compared to an untransformed yeast, increased ability to produce carbon dioxide on fermentation in a maltose-containing medium, wherein both the untransformed yeast and the transformed yeast enhance fermented maltose, characterized in that the transformed yeast contains at least one DNA construct having at least one gene encoding maltospermease, maltase or a maltose regulator protein, and which is expressed in said transformed yeast. A yeast according to claim 1, characterized in that said gene (s) is placed under a transcriptional control which is neither sensitive to glucose repression nor induced under the influence of maltose. Yeast according to claim 2, characterized in that said gene or genes are under transcriptional control of the promoter of alcohol dehydrogenase I (ADHI) and / or translation elongation factor (EFlaA). ____ Yeast according to claim 3, characterized in that the promoters are • derived from a yeast belonging to the family Saccharomces. preferably from the yeast Saccharomyces cerevisiae. Yeast according to any one of claims 1-4, characterized in that said construction forms part of the episomal element. Yeast according to any of claims 1-5, characterized in that said structure is integrated into the chromosome of said yeast. WO 473 Yeast according to any one of claims 1-6, characterized in that it contains at least two of said constructions. Yeast according to any of claims 1-7, characterized in that said yeast is free from heterologous DNA. 9. Transformed yeast belonging to the genus Saccharomces and which increases compared to the untransformed yeast ferment maltos to ethanol and carbon dioxide at a higher rate, wherein both the untransformed yeast and the transformed yeast ferment ferment maltose, characterized in that the transformed yeast contains a DNA construction which is substantially free of prokaryotic DNA, and said DNA construct contains at least one gene encoding maltospermease, maltase or a maltose regulator protein. Yeast according to claim 9, characterized in that maltase is placed under transcriptional gene expression control by the promoter of translation elongation factor (EFlaA). Yeast according to claim 9 or 10, characterized in that maltospermease is placed under transcriptional gene expression control by the promoter of alcohol dehydrogenase I (ADHI). Yeast according to any of claims 9-11, characterized in that said promoters are derived from one: yeast belonging to the genus Saccharomvces. Yeast according to any of claims 1-12, characterized in that it is one of the following yeast strains: Saccharomyces cerevisiae ApGb-iA32 / G418 (Example 2), Saccharomyces cerevisiae ApGb-iRRol (Example 2), 100473 Saccharomyces cerevisiae ApGb eMAL6g (example 1), Saccharomyces cerevisiae ApGb-eMAL61 (example 1), Saccharomyces cerevisiae ApGb-eMAL63 (example 1), Saccharomyces cerevisiae CpGb-eMAL6g (example 1) and Saccharomyces cerevisiae ApGb-p2R Yeast according to any of claims 1-13, characterized in that it is fast dry yeast or active dry yeast with a moisture content of 3-8%. Yeast according to any of claims 1-13, characterized in that it is pressed yeast. Yeast according to claim 15, characterized in that its gas production is at least 340 ml / 285 mg of the yeast dry weight for 165 minutes in sample B and at least 170 ml / 285 mg of the yeast dry weight for 165 minutes in sample B ', and preferably 380 -450 ml / 285 mg of yeast dry weight for 165 minutes in sample B and 180-240 ml / 285 mg of yeast dry weight for 165 minutes in sample B ', and preferably at least 400 ml / 285 mg of yeast dry weight for 165 minutes in sample B and at least 190 ml / 285 mg of the yeast dry weight for 165 minutes in Sample B '. 17. Yeast according to claim 16, characterized in that its gas production is 400-500 ml / 285 mg of the yeast dry weight for 165 minutes in Sample B and preferably 440 ml / 285 mg of the yeast dry weight for 165 minutes in Sample B. Yeast according to claim 16 or 17, characterized in that it is fast dry yeast or active dry yeast with a moisture content of 3-8%. Yeast according to claim 14, characterized in that its gas production is at least 310-360 ml / 285 mg of the yeast dry weight for 165 minutes in sample C and 145-195 ml / 285 mg of the yeast dry weight for 165 minutes in sample C and before - il in »il .; in particular, at least 330 ml / 285 mg of the yeast dry weight for 165 minutes in Sample C and at least 155 ml / 285 mg of the yeast dry weight for 165 minutes in Sample C or 320-400 ml / 285 mg of the yeast dry weight for 165 minutes in sample C and preferably at least 350 ml / 285 mg of the yeast dry weight for 165 minutes in sample C. 20. Vector, characterized in that it has one of the following: pGb-eMAL6g (shown in Fig. 1), pGb-eMAL61 (listed in Fig. 2), pGb-eMAL63 (shown in Fig. 3). , pGb-iA32 / G418 (shown in Fig. 6), pGb-iRRol (listed in Fig. 7), pGb-M6g (A-9) (shown in Fig. 4), pGb-RBRR01 (listed in FIG. 11), pGb-RBN3 (listed in FIG. 10), or pGb-RBREG01. Use of a yeast according to any of claims 1-19 in the production of fermented flour products or alcoholic beverages or other alcoholic products. A process for the preparation of such a transformed yeast belonging to the genus Saccharomces and which has, compared to an untransformed yeast, enhanced the ability to produce carbon dioxide in fermentation in a maltose-containing medium, both fermenting the untransformed yeast and the transforated yeast. maltose, characterized in that a yeast contains at least one DNA construct having at least one gene encoding maltospermeas, maltase or a maltose regulator protein. Method according to claim 22, characterized in that the structure contains at least two said genes. A method according to claim 22, characterized in that the nested gene or said genes are under transcriptional control of the promoter of alcohol dehydrogenase I (ADHI) and / or translation elongation factor (EF10A). 25. A method according to any one of claims 22-24, characterized in that said gene (s) is located (s) under such a transcriptional control that is neither sensitive to glucose repression nor induced under the influence of maltose. Method according to any of claims 22-25, characterized in that said structure forms part of the episomal element. Method according to any of claims 22-25, characterized in that said structure is integrated into the chromosome of said yeast. 28. A method according to any one of claims 22 to 27, characterized in that at least two said DNA constructs are introduced into the yeast. A method according to any of claims 22-25 or 27-28, wherein said yeast is free from heterologous DNA, characterized in that the genes are integrated into the chromosome using gene replacement methods. 30. A method according to claim 24, characterized in that said promoters are derived from a yeast belonging to the genus Saccharomyces. Method according to any of claims 22-30, characterized in that said yeast is Saccharomyces ce-revisiae. 32. Use of a yeast prepared according to anything to ill 1 HU I! ! No. 100473 of claims 22-31 for the preparation of dough or similar products, bread or similar products or alcoholic beverages or other alcoholic products. Use of a yeast prepared according to any of claims 22-31 for the production of an enzyme having maltase activity or maltospermeas activity.