CA1335264C - Yeast strains providing for an enhanced rate of the fermentation of sugars, a process to obtain such yeasts and the use of these yeasts - 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

1 ~ 335264 ~ =

NEW YEAST STRAINS PROVIDING FOR AN ENHANCED RATE OF
THE FERMENTATION OF SUGARS, A PROCESS TO OBTAIN SUCH
YEASTS AND THE USE OF THESE YEASTS

The invention relates to new yeasts capable of im-proving the fermentation of sugars, to a process to construct such yeasts and to the use of these improved yeasts.
It is well known that yeast strains belonging for example to the genus S~cch~romYces 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 exists in several formulations comprising com-pressed yeast or fresh yeast and dried yeast. Dried yeast is 15 available as active dry yeast and as instant dry yeast with moisture contents of about 6-8% and 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, for ex-ample, is dependent on the presence of a specific maltose per-mease. This carrier may exist in two ~orms distinguished by differences in maximal velocity (Vmax) and affinity constant (Km) (A. Busturia and R. Lagunas, Biochim. Biophys. Acta 820, 25 324 (1985)). The translocation of maltose across the yeast plasma membrane is coupled to the electrochemical proton gradient in this membrane. For every maltose molecule taken up one proton is symported (R. Serrano, Eur. J. Biochem. 80, 97 (1977)).
Intracellularly, maltose is hydrolyzed to two molecules of glucose in a reaction catalyzed by maltase (alpha-glucosidase). Glucose is subsequently converted to carbon dioxide and ethanol via the Embden-Meyerhof pathway. In comparison with the fermentation of glucose two additional 7~

- 2 _ t 3352 64 enzymes are required for the fermentation of maltose viz. mal-tose permease and maltase. The synthesis of these enzymes is induced by maltose and repressed by glucose, fructose or man-nose. In non-sugared (l~lean~) doughs maltose is the most abun-5 dant sugar available to yeast. In case sucrose is added to thedough this disaccharide is hydrolyzed extracellularly by yeast to glucose and fructose. Subsequently these hexoses are taken up by yeast by action of distinct permeases.
It is generally found that addition of sucrose to 10 media containing maltose, as for example dough, inhibits the metabolism of maltose by yeast cells. This is due o the fact that transcription of genes encoding maltose permease and maltase is repressed by glucose (R.B. Needleman, D. s. Kaback, R.A. Dubin, E.L. Perkins, N.G. Rosenberg, K.A. Sutherland, D.B.
15 Forrest and C.A. Michels, Proc. Natl. Acad. Sci. USA 81, 2811 (1984)).
Genes required for the uptake and hydrolysis of mal-tose are clustered in a MAL-locus (R.B. Needleman et al. Supra).
Strains of SaccharomYces may contain up to five MAL-loci (MAL 1-20 4 and MAL 6), which are unlinked and located at the telomers ofdifferent chromosomes (J.L. 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 reg-ulator) required for the induction by maltose (R.B. Needleman et 25 al. Supra; J.D. Cohen, M.J. Goldenthal, T. Chow, B. Buchferer and J. Marmur, Mol. Gen. Genet. 200, 1 (1985); R.A. Dubin, E.L.
Perkins, R.B. Needleman and C.A. Michels, Mol. Cell. Biol. 6, 2757 (1986)). Said genes have been isolated and cloned (A.O.
J.D. Cohen et al., supra; R.B. Needleman et al., Supra; ~.J.
30 Federoff, J.D. Cohen, T.R. Eccleshall, R.s. Needleman, s.A.
Buchferer, J. Giacalone and J. Marmur, J. Bacteriol. 149, 1064 (1982)).
As mentioned above yeast fermentation in lean dough depends on maltose as main substrate. Maltose is produced in the dough from starch by action of amylases, which are normally present in the flour. In addition, the flour contains a vari-able amount (0-0.5%) free sugars like glucose, raffinose, etc.
X

~ - 3 - 1335264 (H. Suomalainen, J. Dettwiler and E. Sinda, Process Biochem. 7, 16 (1972)). These sugars are rapidly consumed by the yeast.
Several studies have been published investigating the possible correlation between maltose fermentation and leavening activity 5 of baker's yeast. In some cases a positive correlation was found between the rate of maltose fermentation with activities of maltase and maltose permease. However a positive correlation between the activities of maltase and maltose permease with leavening ability in lean dough could not be observed (P. Hau-10 tera and T. Lovgren, J. Inst. srew. 81, 309 (1975); T. Lovgrenand P. Hautera, Eur. J. Appl. Microbiol. 4, 37 (1977)).
Transformation of yeast cells with multicopy plas-mids containing genes encoding maltase and maltose permease yielded a fourfold increase in specific activity of maltase but 15 maltose permease activity was not enhanced. Introduction of extra genes encoding the regulatory protein did result in a mod-erate increase in specific activity of maltase but again no ef-fect was observed on maltose permease activity (J.D. Cohen et al., Supra). Obtained transformants were not assayed for carbon 20 dioxide or ethanol production by these investigators. In fact prior art discouraged per~orming such tests since it had been shown repeatedly that there was no correlation between activi-ties of maltose permease and maltase with carbon dioxide pro-duction (leavening activity) in lean dough (H. Suomalainen, J.
25 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).
We have now found that yeasts, transformed by in-tegrative plasmids of which examples will be described here-30 after, show an enhanced level of maltose permease and maltaseactivity, compared to the untransformed strain. These enhanced maltase and maltose permease activities surprisingly coincide with an increase of CO2 production or leavening activity, as was also observed in case of yeast transformed with episomal 35 vectors.
These improved yeasts show higher rates of ~-r . ~

- 4 - 1 33 52 6 ~

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 methods provided involve application of recombinant DN~ techniques in such a way that the rate of maltose fermentation by said yeasts is increased drastically irrespective of the presence of other sugars as for example glucose.
Furthermore these improved yeast strains show an excellent leavening activity (gas production) in dough.
Analogously, the higher rate of ethanol production of these yeasts will result in a reduced fermentation time for yeast employed in the production of potable and industrial alcohol or a larger amount of a alcohol produced in a certain time.
Furthermore in case (accumulation of) maltose is inhibiting enzymes, which convert sugar or starch, rapid removal of maltose is advantageous for said fermentation process. By applying the process of the present invention a yeast can be obtained in which (an) extra gene(s) is (are) introduced encoding maltase and/or maltose permease.
The present invention provides a tran~ormed yeast and a process to produce said yeast improved in the fermentation rate of sugars which comprises the introduction into yeast of at least one,preferably homologous, DNA
construct which comprises at least Gne gene in said yeast encoding a protein promoting the uptake and/or initial metabolic conversion of a transported substrate, the gene being capable of expression in said yeast. The rate of fermentation of sugars can be improved during se~eral phases of the leavening, for example an enhanced rate caused by the fermentation of maltose or sucrose.
By homologous DNA is meant DNA originating from the same yeast genus. For example Saccharomyces is transformed with DNA originating from s~romyces~ In this way it is possible to improve already existing properties of the yeast genus, without introducing new properties, which were not present in the genus before. The improvement of fermentation - 4a - 1 335264 rate of sugars may be obtained under aerobic and/or anaerobic conditions. The genes of interest include permeases, particularly maltose permease, saccharidases, particularly maltase, kinases, particularly hexokinases and glucokinase, and the like. This invention may be applied, for example, for a carrier protein required for the uptake of glucose or fructose and hexokinases and glucokinase which catalyze the initial intracellular metabolic conversion of these hexoses to hexose phosphates.
The present invention also provides efficient methods for introduction into the yeasts of at least one homologous DNA construct.

Advantageously the invention may be applied to a construct which comprises at least one gene which encodes a protein which promote the uptake of maltose and the initial metabolic conversion of maltose to glucose. In this way yeasts can be obtained which have several advantages in comparison with the original (host) strains. The benefits of such improved yeast may be found particularly in the improved ethanol and CO2 production. For example when the invention is applied to baker's yeast this will be an enormous advantage for a baker because the baker requires less time or less yeast in order to develop lean dough because of the improved leavening activity of the novel strains.
It will be appreciated that the transformed yeast according to the invention can be used as starting strain in strain improvement procedures other than DNA mediated transformation, for instance, protoplast fusion, mass mating and mutation. The resulting strains are considered to form part of the invention.
According to a preferred embodiment of the invention the novel strains will consume substantial amounts of maltose in the presence of glucose. Therefc~e bakers may save on sugar expenses as well, since less sugar needs to be added to obtain sweet doughs.
It is well-known that osmotolerant yeasts show a poor performance ~leavening activity) in lean dough. By osmotolerant yeasts is meant yeasts which have a good performance in sweet doughs. Doughs for sweet bakery goods will contain for example 10-30% sugar ~based on flour weight).
Therefore, when an osmotolerant strain is chosen as a host to be t ~ sformed according to the invention an osmotolerant yeast is obtained which not only is applicable in sweet doughs but may also be used in lean dough since its capacity to ferment maltose is improved according to the invention. As a consequence the baker conveniently requires only one type of yeast both for sweet doughs and for lean doughs.
The need for yeast strains that have a good ~ - 5a - 1 33526~

performance in lean as well as in sweet doughs is for example described in U.S. patent No. 4,643,901 and in U.S.
patent No. 4,318,929. To obtain yeast strains which have a good performance in sugar rich and lean dough, U.S.
patent No. 4,643,901 describes a protoplast fusion method:
U.S. patent No. 4,318,929 describes in order to obtain such yeast strains a selection method of strains from a population of diploid strains prepared by hybridization or mutation methods. Both procedures need a lot of experimentation and the results are unpredictable and not reproducible. By using the process of the present invention, controlled and reproducible re~ults with the transformed yeast strains can be obtained. The testing of the strains produced according to the invention can be minimal because the 5 properties of the strain itself are substantially not altered except for the improved properties a~ disclosed.
The pre~ent invention provides a compressed yea~t which shows a gas production Or at least 340 ml/285 mg dry weight of yeast in 165 minutes in Test B and a gas production 10 of at least 170 ml/285 mg dry weight of yeast in Test B'.
Tests ~ and B' are described hereinafter. Preferably the compressed yeast shows a gas production of 380-450 ml/285 mg dry weight of yea~t in 165 minutes in Test B and 180-240 ml/285 mg dry weight of yeast in Test B' and more 15 preferably at least 400 ml/285 mg dry weight of yeast in Test B and at least 190 ml/285 mg dry weight of yeast in Test B', respectively. Advantageously instant dry or active dry yeast is prepared from this compressed yeast. During drying of the compressed yeast generally 15-25% of the leavening 20 activity based on dry matter is lost. The present invention also provides a dried yeast (3-8 wt% moisture) which shows a gasproduction of 310-360 ml/285 mg dry weight of yeast in 165 minutes in Test C and 145-195 ml/285 mg dry weight of yeast in Test C' and preferably at least 330 ml/285 mg dry weight of 25 yeast in Test C and at least 155 ml/285 mg dry weight of yeast in Test C', respectively. The gas values obtained with yeast prepared according to the invention have never been found even when commercially available strains were tested in Tests C and C'. The invention can also be applied for yeasts which show a 30 good performance in leavening activity in the range of 0-6 or 0-10% sugar dough. In this way it is possible to prepare compressed yeasts which show a gas production of 400-500 ml/285 mg dry weight of yeast in 165 minutes in Test B, preferably these compressed yea~ts show a gas production of at 35 least 440 ml/285 mg dry ~elght of yeast. Dried yeasts can then be obtained which show a ga~ production of 320-400 ml/285 mg - 7 - l 335264 dry weight of yeast in 165 minutes in Test C. preferably the dried yeast shows a gas production of at least 350 ml/285 mg dry weight of yeast.
Similar advantages of these novel strains are found 5 in fermentations for the production of potable and industrial alcohol.
Since the overall metabolic rate of these novel strains has been increased when maltose serves as a substrate the overall production rate of metabolites such as glycerol and 10 aroma compounds will be increased as well.
In one aspect of the invention vectors are provided bearing a DNA construct encoding one or more proteins involved in maltose fermentation. The present invention also provides a microbial host, preferably a yeast, which is, for example, a 15 species of Saccharomvces transformed with vectors disclosed by the invention. These vectors may be self-replicating and con-tain advantageously a gene, or combinations of genes, selected from those encoding maltose permease, maltase and maltose regu-latory protein. Surprisingly, it has been found that yeast, 20 transformed with such vectors, shows an enhanced rate of maltose fermentation, which re~ults in an increased rate of co2 produc-tion in dough. These additional genes are located on episomes, however, and it is known from literature that such extrachromo-somal molecules are easily lost during non-selective propagation 25 (i.e. growth in the absence of G418 in this particular case) tC.D. Hollenberg (1982) Gene Cloning 12, Organisms other than E.
S~Q~, Eds. P.H. Hofschneider, W. Goebel, Springer Verlag, 119;
S.A. Parent, C.M. Fenimone, and K.A. sostian (1985), Yeast 1, 83). From a practical point of view it is preferred to culti-30 vate yeast non-selectively and therefore a set of integrating plasmids containing, preferably altered, maltase and/or maltose permease genes are advantageously constructed. By ~altered" is meant the exchange of the natural promoter by another promoter, preferably homologous. In case processes are developed which 35 allow stable proliferation of plasmids in the absence of a sel-ective pressure, integration of newly introduced DNA is not a prerequisite anymore in order to obtain stable transformants.
It is known from literature that one cell contains between 20-100 molecules of these extra plasmids (C.D. Hollen-berg (1982), Supra; A. Takagi, E.N. Chun, C. Boorchird, S.
5 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 level of expression of episomal genes may be in-creased even further by exchange of the original promoters by 10 stronger promoters. It seems likely that in the future it will be possible to allow stable replication of plasmids in the ab-sence of a selective pressure.
The maltase and/or maltose permease genes are ac-cording to the invention advantageously integrated into the 15 yeast chromosome via transformation with linear plasmids (see T.L. Orr-Weaver, J.W. Szostak, R. Rothstein (1981) Proc. Natl.
Acad. Sci. U.S.A. 78, 6354). The obtained yeasts are stable transformants, i.e. altered maltase and/or maltose permease genes can be maintained in the genome even in the absence of 20 selective pressure.
It is known, that during integration only one or a few copies of genes located on a plasmid become integrated into the chromosome. Therefore, in order to obtain similar improve-ments in CO2 production as obtained when episomal vectors are 25 used, the level of gene expression may be advantageously altered by application of strong constitutive promoters, not sensitive to glucose repression. Such promoters are preferred in order to compensate for the difference in gene copy number between yeast transformed with episomal and with integrative vectors, and in 30 order to prevent the effects of glucose repression. It is obs-erved, for instance, that during the first 30-40 minutes of fer-mentation in media containing maltose as main carbon and energy source and relatively low concentrations of glucose, mRNA lev-els of maltose permease and maltase decline to barely detectable levels. Once the glucose has been consumed, induction of gene expression by maltose results in a rapid increase in both mRNA
levels.
As indicated, the genes may be used with their natu-5 ral or wild-type promoter or the promoter may be substituted with a different promoter, preferably a homologous promoter.
Particularly, where the wild-type promoter is regulatable or in-ducible, it may be desirable to provide for constitutive trans-cription, or a stronger or weaker promoter. Conversely, where the wild-type promoter is constitutive, it may be of interest to provide for a regulatable or inducible promoter or a stronger or weaker promoter.
Desirably, strong promoters will be employed, part-icularly where there may be a relatively low copy number of the construct in the host. Strong promoters will normally be those involved with the production of proteins produced at a high lev-el during the life cycle of the yeast or where regulatable, at some period of interest in the life cycle of the yeast, as rela-ted to the subject invention.
Promoters associated with the glycolytic cycle of yeast are of particular interest, which include alcohol dehyd-rogenase I and II, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase, triose phosphate isomerase, glyceraldehydephos-phate dehydrogenase, phosphoglycerate kinase, enolase, phospho-25 glyceromutase, pyruvate kinase, and lactate dehydrogenase.
Other promoters involved with proteins produced in high amount, include promoters associated with ribosomal expression, such as promoters for the transcription of initiation factors, elonga-tion factors, and the like. Particular elongation factors in-30 clude EF-l and EF-2, etc.
Of particular interest is the use of promoters in combination with structural genes involved with maltose metabo-lism, particularly maltase, maltose permease and MAL-regulator.
These promoters may be constitutive or regulatable, so long as the promoter is induced during the fermentation of the sugar.
For example, many of the glycolytic promoters are activated in ~ 1 335264 the presence of a sugar or a sugar metabolite, such as ethanol.
Thus, promoters such as alcohol dehydrogenase will be active during the leavening of flour. Similarly, those promoters as-sociated with cell proliferation will also be active during the leavening of dough. Furthermore, by providing for promoters which are not regulated by the MAL regulator, the yeast may be used in the presence of glucose, without repression. In addi-tion, the genes do not require maltose for induction.
Where the wild-type promoters are employed in con-10 junction with the structural genes of interest, it may be de-sirable to provide for enhanced production of a regulatory pro-tein. In this way, the regulatory protein may be maintained at a high level, when the inducer is present. For example, in the presence of maltose, the MAL regulatory protein will be ex-15 pressed at a high level, so as to provide for expression of theother proteins associated with maltose metabolism and regulated by the MAL regulatory protein.
The alterations in gene expression, as described in detail in the experimental procedures, comprise the exchange of 20 the original promoters plus (part of) the untranslated leader sequences ~or those o~ alcohol dehydrogenase I (ADHI ) and trans-lation elongation factor EFlaA preferably derived from the host yeast, for example S~ccharomYces. AS a consequence, expression will become insensitive to glucose repression and independent of 25 maltose for induction.
It has been found that yeasts, transformed by these integrative plasmids show an enhanced level of maltose permease and maltase activity compared to the untransformed strain.
These enhanced maltase and maltose permease activities surpris-ingly coincide with an increase of CO2 production or leaveningactivity, as was also observed in case of yeast transformed with episomal vectors.
The obtained improvement in leavening activity is maintained during storage even at elevated temperatures, for 35 example, at 20-25C. The relative loss of leavening activity during storage is virtually identical for the parental strains and the strains according to the invention. The leavening ac-tivity in high sugar doughs is not affected by the introduced modifications since the leavening activity of the novel strains 5 transformed with integrative plasmids is as good as that ob-tained with the host strain.
The subject yeast host will have at least one copy of the construct, any may have two or more, usually not exceed-ing about 200, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers. Integration or non-inte-gration may be selected, depending upon the stability required for maintenance of the extrachromosomal element prepared, the number of copies desired, the level of transcription available 15 depending upon copy number, and the like.
The construct may include one or more structural genes with the same or different promoters. The construct may be prepared in conventional ways, by isolating the desired genes from an appropriate host, by synthesizing all or a portion of =~
20 the genes, or combinations thereof. Similarly, the regulatory signals, the transcriptional and translational initiation and termination regions, may be isolated from a natural source, be synthesized, or combinations thereof. The various fragments may be subjected to endonuclease digestion (restriction), ligation, sequencing, ln vitro mutagenesis, primer repair, or the like.
The various manipulations are well known in the literature and will be employed to achieve specific purposes.
The various fragments may be combined, cloned, iso-lated and sequenced in accordance with conventional ways. Af-30 ter each manipulation, the DNA fragment or combination of frag-ments may be inserted into the cloning vector, the vector trans-formed into a cloning host, e.g. ~. coli, the cloning host grown up, lysed, the plasmid isolated and the fragment analyzed by re-striction analysis, sequencing, combinations thereof, or the like.
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- 12 - l 3352 64 Various vectors may be employed during the course of development of the construct and transformation of the host cell. These vectors may include cloning vectors, expression vectors, and vectors providing for integration into the host or the use of bare DNA for transformation and integration.
The cloning vector will be characterized, for the most part, by having a replication origin 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, ease of se-quencing, excision, or the like. In addition, shuttle vectors may be employed, where the vector may have two or more origins of replication, which allows the vector to be replicated in more than one host, e.g. a prokaryotic host and a eukaryotic host.
Expression vectors will usually provide for inser-tion of a construct which includes the transcriptional and translational initiation region and termination region or the construct may lack one or both of the regulatory regions, which will be provided by the expression vector upon insertion of the 20 sequence encoding the protein product. Thus, the construct may be inserted into a gene having unctional transcriptional and translational 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. Mor-25 mally, it would be desirable for the initiation codon to be 5'of the existing initiation codon, unless a fused product is ac-ceptable, or the initiation codon is out of phase with the ex-isting initiation codon. In other instances, expression vectors exist which have one or more restriction sites between the init-iation and termination regulatory regions, so that the structu-ral gene may be inserted at the restriction site(s) and be under the regulatory control of these regions. Of particular interest for the subject invention as the vector for expression, either for extrachromosomal stable maintenance or integration, are ~ - 13 - l 3 3 5 2 6 ~

constructs and vectors which in their stable form in the host are free of heterologous (non-Saccharomyces) DNA.
According to a further aspect of the invention processes are provided to produce yeasts which exhibit all the advantages described above whereas in addition prokaryotic DNA
sequences have been removed. This has been accomplished by gene replacement techniques (R.J. Rothstein (1983) Methods in Enzymology, 101, 202). According to the present invention these techniques are now advan~ageously applied to ,~h~romyces cells. For example, transformation of Saccharomyces cells, with a vector contA; n; ng 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). ~fter introduction of this DNA into a Saccharomyces host cell homologous recombination of the newly introduced DNA takes place with the chromosomal sporulation-specific gene. As a conse~uence altered maltase and/or maltose permease genes embedded between the sporulation specific sequences become integrated into the chromosome. Resulting transformants are completely devoid of prokar~otic DNA.
It will be appreciated to realise that even better results may be obtained if the optimal ratio of maltase and maltose permease activity is determined. This can be done by varying the promoters of both genes or by integration of different numbers of ~altered) maltase and maltose permease genes into the yeast genome, for example, by using several integration loci, according to methods as described below.
The optimal ratio of maltase and maltose permease activity can also be obtained using maltase or maltose permease genes encoding other isoenzymes of maltase and maltose permease.
Furthermore any enzyme can be applied, having at least maltase or maltose permease activity. In addition, genes encoding the MAL-regulatory protein can be integrated into the genome in an analogous way (see also Example 1), either under control of its own, natural promoter or under control of another promoter, ~ S - 13a - 1 335264 pre~erabl~ Saccharomyces. This can also be useful in order to obtain in optimal ratio of ma~tase and maltose permease activity.

- 14 - l 3 3 5264 List o~ deposited strains The following strains have been deposited with the Centraal Bureau voor Schlmmelcultures~ Baarn, Holland:

SaccharomYces cerevisiae 237 Ng (strain A) has been deposited with the CBS under the accession number 158.86 on March 25, 1986;
SaccharomYces cerevisiae DS 15543 (strain C) has 10 been deposited with the CBS under the accession number 406.87 on September 3, 1987;
F.scherichia coli harbouring plasmid p21-40 has been deposited with the CBS under the accession number 400.87 on August 28, 1987;
Escherichia coli harbouring plasmid pYEF46 has been deposited with the CBS under the accession number 401.87 on August 28, 1987;
~ scherichia coli harbouring plasmid pY6 has been deposited with the CBS under the accession number 402.87 on 20 August 28, 1987;
~ .scherichia coli harbouring plasmid peG418 has been deposited with the CBS under the accession number 160.86 on March 25, 1986;
Escherichia coli harbouring plasmid pTZ19R has been 25 deposited with the CBS under the accession number 405.87 on September 3, 1987;
Escherichia coli harbouring plasmid pTZ19R/ADHI has been deposited with the CBS under the accession number 404.87 on September 3, 1987;
Escherichia coli harbouring plasmid pl53-215 AK has been deposited with the CBS under the accession number 403.87 on September 3, 1987.
Escherichia coli harbouring plasmid pLF24 has been deposited with the CBS under the accession number 156.88 on 35 March 8, 1988.

~r ~ - 15 - l 3 3 5 2 6 4 Fscherichia ~Qli harbouring plasmid pUT332 has been deposited with the CsS under the accession number 158.88 on March 8, 1988.
Fscherichia coli harbouring plasmid pTZ18R has been 5 depo~ited with the CsS under the 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 of indi-cated genes. Plasmids are drawn schematically and not to scale.
Abbreviations: G418, Tn5 gene (under control of ADHI promoter) conferring resistance to G418; P, PvuI; X, XbaI; S, SalI; H, 15 HindIII; CIP: calf intestine phosphatase.

Figure 2 describes the construction of pGb-eMAL61.
Plasmids are drawn schematically and not to scale. Abbrevia-tions: ~maltase partial deletion maltase gene; B, sglII. See 20 also description of Fig. 1.

Figure 3 describes the construction of plasmid pGb-eMAL63. Plasmids are drawn schematically and not to scale.
Abbreviations: (K), filled-in KpnI site; (S), filled-in SalI
site; Klenow, large subunit DNA polymerase; H, HindIII. See also description of Fig. 1.

Figure 4 describes the construction of pGb-M6g (~-9). Abbreviations: H, HindIII; St, StuI; Klenow, large frag-30 ment DNA polymerase I; EV, EcoRV; Xh, XhoI; M, MluI; fl ori,origin of replication phage ~1; amp, ampicillin resistance gene;
s.p. sequence primer. Arrows indicate 5' ~ 3~ direction. Del-eted area is indicated with dotted lines and ~.

~ 1 335264 Figure 5 describes the sequence of plasmid pGb-M6g (~-9 ) .
a) maltase and maltose permease genes. Arrow indi-cates direction of transcription. St (StuI) served as start-5 point for construction of deletion mutant. Relevant parts ofsequence of intergenic area are shown below this map.
b) sequence of pGb-M6g (~-9). Deleted area extends from -9 to -417. Polylinker refers to the oligonucleotides which have been ligated onto the Bal31-treated DNA (see also 10 Fig. 4).

Figure 6 describes the construction of plasmid pGb-iA32/G418. Arrows indicate direction of transcription. Plas-mids are drawn schematically and not to scale. Abbreviations:
15 H, HindIII; EV, EcoRV; fl ori, origin of replication phage fl;
amp, ampicillin resistance gene; G418, Tn5 gene (ADHI promoter) conferring resistance to G418; S, SmaI; Hc, HincII; pADHI, pro-moter alcohol dehydrogenase I gene + part 5'leader (hatched area).
Figure 7 describes the construction o~ plasmid pGb-iRR01 a) plasmid pT4 is not drawn to scale. Abbrevia-tions: E, EcoRI; B/Bg, BamHI/BglII ligation; H, HindIII
b) mutagenesis on pT4 in order to fuse the EFl~A
promoter + 5'leader to the five N-terminal amino-acids codons of the maltase gene in such a way that a BglII site is created as well. Relevant sequences are shown.
- Mutagenesis primer is partly complementary (indi-30 cated with dots) to the EFlaA sequence. In the region of mis-matches are the maltase codons and the -boxed- BglII recognition site (note that the presented orientation of the mutagenesis primer is 3l ~ 5l, i.e. the BglII site should be read from right to left (5' ~ 3').
- In the maltase sequence the M-terminal five amino acid codons are indicated. The BclI recognition site is boxed.
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~ - 17 - l 3352 6 4 - In the sequence of pT4-M, the sequence covering the mutation is shown. BglII site is boxed. Asterisks indicate the deviation from the maltase nucleotide sequence. The devia-tion in the fourth codon is a silent mutation.
c) plasmids are not drawn to scale. Abbreviations:
H, HindIII; Bc, BclI; E. EcoRI; Bg, BglII; EV, EcoRV; Bg/Bc, BglII/BclI ligation; pEFlaA (hatched box), 5I flank (promoter +
5~leader sequence) of EFlaA; fl ori, origin of replication of phage fl; amp, ampicillin resistance gene.
d) plasmids are not drawn to scale. Abbreviations:
see c).

Figure 8 describes the construction of plasmid pGb-SNENS. Plasmids are drawn schematically and not to scale. Ab-15 breviations: E. EcoRI; H, HindIII; Sf, SfiI; N, NotI; fl ori, origin of replication phage fl; amp, ampicillin resistance gene.
Arrows indicate 5' ~ 3I direction. Oligo 3 and 4 are synthetic oligodeoxynucleotides with the base sequence as indicated.

Figure 9 describes the construction of plasmid pGb-RB2. Plasmids are drawn schematically and not to scale. Ab-breviations: E, EcoRI; Bg, BglII; H, HindIII; Hc, HincII; B.
BamHI; Sm, SmaI; P, PstI; fl ori, origin of replication - phase fl; amp, ampicillin resistance gene. Arrows indicate 5' ~ 3' 25 direction. Oligo 5 and 6 are synthetic oligodeoxynucleotides with the base sequence as indicated. Underlined are the TAA
translational stopcodons in all reading frames.

Figure 10 describes the construction of plasmid pGb-30 RBN3. Arrows indicate direction of transcription. Plasmids aredrawn schematically and not to scale. Abbreviations: Sf, SfiI;
Sm, ~maI; H, HindIII; fl ori, origin of replication phage fl;
amp, ampicillin resistance gene; G418, Tn5 gene (under control of ADHI promoter) conferring resistance to G418. Ev, EcoRV; Hc, 35 HincII.

- 18 - l 335264 Figure 11 describes the construction of plasmid pGb-RBRR01. Plasmid pGb-iRR01 is fully described in Figure 7 and contains the maltase gene under direction of EFlaA promoter (pEFl~A) and the maltose permease gene under direction of the 5 alcohol dehydrogenase I promoter (pADHI) (both promoters are indicated with hatched boxes). pGb-RB2 is described in Figure 9. In pGb-RBRR01 the SIT4 containing fragment is divided into two parts ("SIT4 flanks~'). Plasmids are drawn schematically and not to scale. Abbreviations are as in legends to Figure 10.
10 Arrows indicate direction of transcription.

Figure 12 describes the one-step gene disruption.
In step 1, pGb-RBN3 is digested with SfiI. ThiS liberates a DNA
fragment which as on both sides homology to the SIT4 gene reg-ion. This directs integration to the SIT4 gene (see also R.J.Rothstein (1983) in Methods of Enzymology, 101, 202). The SIT4 gene region is indicated by a hatched box. Both chromosomal al-leles have been shown schematically. In step 2 the resulting strain ApGb-RBN3 is transformed with both pUT332 (undigested) 20 and pGb-RBRR01 digested with SfiI. The principle of cotransfor-mation is well documented (c~. A.H. srand~ I. sreeden, J. Abra-ham, 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 we have used resistance against phleomycin (plasmid pUT332) but of course 2~-derived episomal plasmids con-ferring resistance to other antibiotics like hygromycin B can be used as well. pUT332 and phleomycin are commercially available from Cayla, Avenue Larrien, Centre Commercial de Gros, 31094 Toulouse Cédex, France. On pUT332, the phleomycin-resistance 30 gene is derived from transposon Tn5 and has been placed under the direction of a yeast promoter. In step 3 the episomal plasmid pUT332 is removed from the transformants by growth in non-selective medium (curing). Abbreviations: G418r, G418 resistance gene under direction of ADHI promoter; Sf, end of a 35 DNA fragment generated by SfiI digestion; MAL, an altered mal-tase and maltose permease gene. See also Fig. 9 and 11 for de-tails of the plasmids, +pUT332, episomal plasmid pUT332.

Figure 13 describes the correlation between the in-5 crease in specific activity of maltose permease and maltase withthe disappearance of glucose from medium A. Graphs are typical for commercial baker's yeast strains as for example strain A.

Figure 14 describes the specific activities of mal-tose permease in strain A and its rDNA derivatives during a sim-ulation of dough-rise in medium A.

Figure 15 describes the specific activities of ma-ltase during a simulation of dough-rise in strain A and its rDNA
15 derivatives in medium A containing maltose as main carbon and energy source.

Figure 16 describes the fermentation of maltose dur-ing a simulation of dough-rise by strain A and its rDNA deriva-20 tives in medium A containing maltose as main carbon and energy ~:ource .

Figure 17 describes the fermentation of maltose dur-ing a simulation of dough-rise by strain A and its rDNA deriva-tives in medium B containing glucose as main carbon and energysource.

The following experimental data are given to illus-trate the invention. It has to be understood that a person skilled in the art who is familiar with the methods may use other yeast strains and vectors which can be equally used for the purpose of the present invention. These alterations are included in the scope of the invention.

~r Clonin~ techni~ues For general cloning techniques re~erence is made to the handbook of Maniatis et al. (T. Maniatis, E.F. Fritsch, J.
5 Sambrook (1982) Molecular Cloning, A Laboratory Manual). Res-triction enzymes are used as recommended by the manufacturer and are obtained either from New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Boehringer Mannheim (Boehringer).
In general 1 to 5 units of enzyme are needed to cleave 1 ~g of 10 DNA.
Transformation of E. coli was carried out using the CaCl2-technique (T. Maniatis et al., Supra).

Const~uction of recombinant ~lasmids 1. pGb-eMAL6g This plasmid is capable of self-replicating in yeast and contains the genes encoding maltose permease and maltase.
Its construction is outlined in Fig. 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 resistance to G418 under direction of the promoter alcohol dehydrogenase I (ADHI) from yeast, similar to that as described by Bennetzen and Hall (J.C. Bennetzen and B.D. Hall (1982) J. Biol. Chem. ~, 3018). peG418 was cleaved with HindIII, dephosphorylated with CIP and ligated with a di-gest of pY6 x HindIII x PvuI. pY6 is described (R.B. Needleman and C. Michels (1983) Mol. Cell. Biol. 3, 796; R.B. Needleman, 30 D.B. Kaback, R.A. Dubin, S.L. Perkins, N.G. Rosenberg, K.A.
Sutherland, D.B, Forrest, C. Michels (1984) Proc. Natl. Acad.
Sci. U.S.A. 81, 2811) and contains a 7.0 kb HindIII fragment comprising the MAL6g locus. This yielded pGb-eMAL6g.

. ~

- 21 - l 335264 2. pGb-eMAL61 This 2~-derived episomal plasmid contains the gene encoding maltose permease. Its construction is outlined in Fig.
5 2.
pGb-eMAL6g contains two BglII sites, both lying in the maltase gene. This 1.4 kb BglII has been deleted from pGb-eMAL6g by digestion with BglII, followed by dilute religation to promote intramolecular ligation. Such a deletion has been shown 10 to destroy maltase unction (J.D. Cohen, M.J. Goldenthal, T.
Chow, B. Buchferer and J. Marmur (1985) Mol. Gen. Genet. 2Q, 1).

3. pGb-eMAL63 This 2~-derived episomal plasmid contains DNA cover-ing the MALp function (regulatory protein gene or MAL-regula-tor). Its construction is outlined in Fig. 3.
From p21-40 (R.B. Needleman and C. Michels (1983), Supra) the KpnI-SalI fragment was isolated containing the regu-latory protein gene. This fragment was made blunt-ended using T4 DNA polymerase and the Klenow-DNA polymerase and therea~ter cloned into the filled-in HindIII site of peG418.

4. pGb-M6g(~-9) This plasmid is a promoter-deletion mutant made in the intergenic region of the divergently transcribed genes mal-tose permease and maltase (see Fig. 4). This region contains the promoters for both genes (S.H. Hong and J. Marmur (1986) 30 Gene 41, 75). This deletion mutant has been made in order to replace the original promoters. The construction comprises the following steps:

a) the approximately 7.0 kb HindIII fragment con-35 t~ln;ng the genes for maltase and maltose permease (see also - 22 _ l 33 5264 Fig. 1) was cloned into the HindIII site of pTZ19R. This plas-mid is commercially available (Pharmacia). This results in pGb-M6g.

b) pGb-M6g was linearized with StuI, which cuts in the intergenic region. The StuI-generated ends served as star-ting point for the exonuclease Bal31 in order to nibble off (parts of) the promoters-cont~;n;ng intergenic area. StuI lies closer to the maltose permease gene (S.H. Hong and J. Marmur (1986), Supra). The Bal31 incubation was carried out as des-cribed by Maniatis et al (T . Maniatis, E.F. Fritsch and J. Sam-brook (1982), Supra). At appropriate times samples have been removed from the reaction and incubated with Klenow DNA polymer-ase to make blunt-ends. Then synthetic linkers have been lig-15 ated onto the ends containing several restriction sites.
The following complementary oligodeoxynucleotides have been used:

1. 5~ GATATC CTCGAG AGGCCT A 3' 2. 3~ CTATAG GAGCTC TCCGGA TGCGC 5~

In double-strand form restriction sites are created for EcoRV
(GATATC), XhoI (CTCGAG), Stui (AGGCCT), ligation at the sticky 25 end creates a MluI site (ACGCGT). After kinase-reaction, the linkers have been 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 chromato-graphed through a 5 ml Sepharose Cl-2B column in order to sepa-30 rate the non-ligated oligodeoxynucleotides from the DNA frag-ment. Fractions containing this linear DNA were pooled, ligated to plasmid DNA and introduced into bacteria.

c) the resulting set of deletion mutants were sub-35 jected to sequence analysis. To this end, the double-stranded plasmids were converted into single-stranded DNA by superinfec-tion with a helper phage (protocol according to recommendation of supplier). The single-stranded templates were extracted by normal M13 procedures for use in dideoxysequencing (F. Sanger, 5 S. Nicklen and A.R. Coulson (1977) Proc. Natl. Acad. Sci. 74, 5463). AS a primer we have used a synthetic oligodeoxynucleo-tide (5'-GAATTCGGTAGCGTTCACGC-3'), complementary to a stretch of DNA near the ATG startcodon of the maltose permease gene. Its orientation is such that the sequence is read towards the pro-10 moter (see also Fig. 4).
The deletion mutant in which most of the maltosepermease promoter had been removed, was selected for further experiments. (Part of) the maltase promoter is still present (note that the StuI site as startpoint for exonuclease treat-15 ment is located asymmetrically in the intergenic region). Fig.5 lists the sequence at the deletion point of the mutant pGb-M6g(~-9). This is compared to the recently determined sequence of this entire area (S.H. Hong and J. Marmur (1986), Supra). In the wild type sequence, one difference has been observed with 20 the published sequence: the C at -878 (numbering according to S.H. Hong and J. Marmur (1986), Supra) is not pre~ent in our sequence. In accordance, the DNA cannot be digested with HpaI
or HincII at this position.
Plasmid pGb-M6g(~-9) is the starting plasmid to fuse 25 other promoters to both the maltose permease gene and the mal-tase gene (see below).

5. pGb-iA32/G418 This plasmid is an integrating yeast plasmid. It contains the maltose permease gene, hooked onto the alcohol de-hydrogenase I promoter and part of its 5'leader sequence. Its construction was as follows (Fig. 6):

a) plasmid pTZ19R/ADHI contains a 1.4 kb BamHI frag-ment with the ADHI promoter, starting at position -15 relative - 24 _ l 335~64 to the AUG codon (J.L. Bennetzen and B.D. Hall (1982) J. Biol.
Chem. ~, 3018). From this plasmid, the 700 bp EcoRV-HincII
fragment has been isolated and ligated to EcoRV digested pGb-M6g(~-9). Resulting plasmids were analyzed with restriction 5 enzyme digestions and the proper orientation was confirmed via dideoxy sequence analysis on single-stranded templates (see Fig.

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-10 HincII) is present between the maltose permease gene and theADHI promoter. Structure and sequence of pGB-A32 is shown in Fig. 6a.

b) plasmid pGb-A32 was provided with the dominant selection marker G418reS. A 1.9 kb EcoRV/HincII fragment con-tains the Tn5 gene conferring resistance to G418 under direction of the promoter alcohol dehydrogenase I. This fragment was iso-lated by an EcoRV/HincII double digestion of plasmid 153-215 AK.
The EcoRV/HincII fragment was cloned into the SmaI site of pGb-20 A32. This yielded pGb-iA32/G418.

6. pGb-iRR01 This plasmid is an integrating plasmid. It contains the maltose permease gene under direction of the promoter alco-hol dehydrogenase I and the maltase gene under direction of the promoter translation elongation factor EFl~A. The cloning path-way is depicted in Fig. 7. The approach was as follows: the maltase gene contains a BclI site around the fifth amino acid 30 codon. Therefore the EFl~A coding region was mutagenized in such a way that the first five amino acids became identical to those of the maltase protein. A BglII site was co-introduced at the position of the BclI site. Conversion of a BclI site to a BglII site is a silent mutation in the fourth codon. Via a 35 sglII/sclI ligation the EFl~A promoter and leader se~uence could be fused to the rest of the maltase gene. The procedure com-prised the following steps:

q,.,~

- 25 - l 3 3 52 6 4 a) the starting plasmid was pYEF46 (S. Magata, K.
Nagashima, Y. Tsunetsugu-Yokota, K. Fuyimura, M. Miyaraki and Y.
Kaziro (1984) EMBO J. 3, 1825) which contains the entire gene coding for EFlaA. A 2.5 kb BglII fragment covering this gene, 5 was isolated and cloned into the BamHI site of pTZ19R. Clone pT4 was picked up (see Fig. 7a) and used for the oligodeoxynuc-leotide directed mutagenesis.

b) after superinfection with helper phage, single stranded (ss) DNA of pT4 was isolated. 400 ng ss DNA, 400 ng heat-denatured pTZ19R x BamHI and 100 ng mutagenesis oligodeoxy-nucleotide (see Fig. 7b) were incubated in a volume of 10 ~1 o 7 mM Tris HCl pH 7.5; 50 mM NaCl and 7 mM MgC12 for 10 minutes at 56C. After 10 minutes at room temperature, second strand synthesis and ligation were started by addition of 1 ~1 of Kle-now DNA polymerase (2U). 1 ~1 of T4 DNA ligase (4U), 1 ~1 TMD
(200 mM Tris HCl pH 7.5, 100 mM MgC12, 100 mM DTT), 4 ~l 2.5 mM
dNTP-mix, 1 ~1 10 mM ATP and 2 ~l H2O. End-volume was 20 ~1, incubation was performed for 16 hours at 17C, after which the 20 mixture was transformed to E. coli JM101. Mutants were screened by colony hybridization with a kinased oligodeoxynucleotide, specific for the mutant (see Fig. 7b). This screening oligomer 5'-GACTATTTCAGATCTTC-3' was complementary to the introduced mal-tase codons and flanking nucleotides. The hybridization was carried out for 16 hours in 6 x NET (1 x NET = 0.15 M NaCl, 0.015 M Tris HCl pH 7.5, 0.001 M EDTA) at 25C. Post-hybridi-zation washes were performed in the same mix at the same temp-erature (3 times 10 minutes), followed by a wash in 3 x NET at 25C. Several positive colonies were analyzed further. BglII, 30 digestion confirmed the presence of a BglII site. In addition, single-stranded DNA was isolated from a BglII-site containing mutant (pT4-M) and subjected to dideoxy sequence analysis using a synthetic 17-mer primer, complementary to nucleotides 87-103 of the EFlaA gene (S. Nagata, K. Nagashima, Y. Tsunetsugu-35 Yokota, K. Fuyimura, M. Miyaraki and Y. Kaziro (1984) EMBO J. 3, '.f~,.

1825) (5'-CAATACCACCACACTTG-3'). The sequence obtained con-firmed the successful introduction of the desired mutations.

c) the next step was to isolate the EFl~A promo-5 ter/NH2-terminal maltase gene segment from pT4-M and to fuse it via BglII/BclI sticky end ligation to the rest of the maltase gene. This step restores the maltase coding region downstream of the EFlaA promoter and leader sequence (see Fig. 7c). Plas-mid pGb-M6g(~-9) was transformed to GM113, an E. coli dam strain, in order to be able to use the BclI site (which is me-thylation sensitive) (GM113: thr~~ leuB6, proA2, tris-4, metBl, lacYl, galK2, ara-14, tsx33, thi-l, thyA12, deoB16, supE44, rpsL260, dam 3). A 3.8 kb BclI/HindIII fragment was isolated, containing the maltase gene except for the very MH2-terminal end. pT4-M was digested with BglII/HindIII and the large 4.1 kb fragment isolated (EFlaA promoter/NH2-terminal and maltase gene). Both fragments were ligated to each other, resulting in pGb-EFMT-3. Sequence analysis confirmed the correctness of all introduced mutations.
d) ~inally, pGb-EFMT-3 was digested with EcoRV and HindIII to purify a 4.8 kbp. EFl~A/maltase promoter/gene frag-ment. From pGb-iA32/G418 (see Fig. 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/G418reS segment. Both were ligated to yield pGb-iRR01 (see Fig. 7d).

7. pGb-RB2 This plasmid serves as cloning vehicle to integrate pieces of DNA into the SIT4 gene of Saccharomvces cerevisiae.
Its construction comprises the following steps (see Figures 8 and 9):

, --., .

- 27 _ l 3 3 5 2 6 4 a) pTZ19R was digested with EcoRI and HindIII to re-place its polylinker by a synthetically made DNA segment of 40 nucleotides. This piece of DNA is made by annealing of the syn-thetic oligodeoxynucleotides 3 and 4 (see Fig. 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 SfiI and in the mid-dle an EcoRI site, as indicated. The resulting plasmid is des-ignated pGb-SNENS.

b) an EcoRI fragment containing the SIT4 gene (Got-tlin-Nin~a and Kaback, vide supra) was isolated from pLF24 (also known having the code pLN420) and cloned into the EcoRI site of 15 pGb-SNENS. This yielded pGb-Spons31. Due to lack of useful re-ference restriction sites, its orientation is unknown.

c) plasmid pGb~Spons31 contains a unique BglII site in the middle of the SIT4 gene. This can be used as site into 20 which any segment of DNA to be transferred to the SIT4 gene by gene replacement, can be cloned. To ~acilitate cloning manipu-lation, the SIT4 gene has been provided with a synthetic piece of DNA, containing several unique restriction sites. The con-struction of pGb-RB2 is outlined in Fig. 9.
The synthetic DNA fragment has two sticky BglII
ends. Its orientation in pGb-RB2, as indicated in Fig. 9, is based on restriction enzyme analysis of pGb-RBM3 (see next).

8. pGb-RBN3 A 1.9 kb EcoRV/HincII fragment containing the G418reS gene under control of the ADHI promoter (see also construction of pGb-iA32/G418), was cloned into the SmaI site of pGb-RB2. This yielded pGb-RBN3 (Fig. 10).

- 28 - l 33 5264 9. pGb-RBRR01 This plasmid contains the maltose permease gene un-der direction of the promoter alcohol dehydrogenase I and the 5 maltase gene under direction of the EFl~A promoter. Both are located on an 8.3 kb HindIII fragment which has been cloned into the SIT4 gene. Its construction is outlined in Fig. 11. To this end, pGb-iRR01 was digested with HindIII and ligated onto pGb-RB2 x HindIII (treated with calf intestine phosphatase).

10 This yielded pGb-RBRR01.

10. pGb-RBREG01 This plasmid contains the MAL-regulator gene under 15 direction of the promoter alcohol dehydrogenase.
I. pGb-RBREG01 can be constructed as follows. From p21-40 (R.B. Needleman and C. Michels (1983J, supra) the SalI
fragment is isolated containing the regulatory protein gene.
This fragment is cloned into the SalI site of pTZ18R. This 20 plasmid is commercially available (Pharmacia). Its orientation is such that the promoter area of the MAL-regulator ~ene is proximal to the T7-promoter sequence of pTZ18R. Using the se-quence-primer of pTZ18R and other newly made oligonucleotide sequence-primer of pTZ18R and other newly made oligonucleotide 25 primers based on the DNA sequence obtained, the promoter area of the MAL-regulator gene and the NH2-terminal encoding part of the gene can be sequenced. This locates the position of the AUG-startcodon. When useful restriction sites are absent in the promoter area, close to the AUG-startcodon, such a site (for in-stance BglII) can be introduced in that area via site-directed mutagenesis with an oligonucleotide according to standard meth-ods (see also construction of recombinant plasmids, section 5b).
After such a mutagenesis the BglII-SalI fragment (containing the MAL-regulator gene without promoter area) and an 35 EcoV-BanHI fragment (cont~;n;ng the ADHI promoter, see also Fig.
7d, pGb-iRR01) can be ligated together into pGb-RB2 (see Fig.
X

- 29 - l 335264 9~, such that the MAl -regulator gene is under control of the ADHI promoter. This will yield plasmid pGb-IRBREG01. Sequence analysis using the dideoxy chain methods with oligonucleotides on ss DNA as template, can easily confirm the correctness of the 5 cloning steps and the orientation of the promoter.

It will be appreciated to recognize that the above-mentioned plasmids constructions merely serve as examples to il-lustrate the invention. Other promoters (preferably Saccharo-10 mYces) can be used (or other parts of the same promoter), otherintegration loci can be selected, other combinations of maltase, maltose permease and MAL-regulator (either under control of their own promoter or under control of another, preferably Sac-charomvces promoter), can be made, using one or more integration 15 sites in the yeast genome. It will ultimately lead to the opti-mal ratio of maltase and maltose permease activity present dur-ing the entire period of anaerobic fermentation.

Yeast ~ransformation Transormation o yeast strains was carried out ac-cording to the method of Ito et al. (H. Ito, Y. Fukuda, K. Mur-ata, A. Kimura (1983), J. Bacteriology 153, 163-168). It in-volves growing Saccharomvces in a standard yeast nutrient medium 25 to a density of 1 to 25, desirably 4 to 10 OD610 nm. The yeast cells are then harvested, washed and pretreated with chaotropic ions particularly the alkali metal ions, lithium, cesium or rub-idium, particularly as the chloride or sulphate, more particu-larly the lithium salts, at concentrations about 2 mM to 1.0 M, 30 preferably about 0.1 M. After incubating the cells for from about 5 to 120 minutes, preferably about 60 minutes, with the chaotropic ion(s), the cells are then incubated with DNA for a short period of time at a moderate temperature, generally from about 20C to 35C for about 5 minutes to 60 minutes. Desir-35 ably, polyethylene glycol is added at a concentration of about25 to 50%, where the entire medium may be diluted by adding an ~ - 30 - l 335264 equal volume of a polyethylene glycol concentrate to result in the desired final concentration. The polyethylene glycol will be of from about 2000 to 8000 daltons, preferably about 4000 to 7000 daltons. Incubation will generally be for a relatively short time, generally from about 5 to 60 minutes. Desirably, the incubation medium is subjected to a heat treatment of from about 1 to 10 minutes at about 35C to 45C, preferably about 42C. For selection of transformants any useful marker may be used, 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. (19800, Nature, 287, 869).
When yeast cells have been transformed with integra-ting plasmids, integration was directed to the MAL-locus using 15 BglII-digested DNA. The integrating plasmids used, contain two BglII sites, both in the maltase gene, 1.4 kb from each other.
This generates double-stranded breaks, which are recombinogenic and stimulate interaction with homologous chromosomal DNA. The gap is repaired from chromosomal information during the integra-20 tion event (T.L. Orr-Weaver, J.W. Szostak, R. Rothstein (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6354). The integration event yields 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 CO2 production experiments, has not been 25 determined.

A scheme was developed in order to obtain stable yeast transformants which do not contain heterologous DNA. Ex-amples of heterologous DNA are the pTZ19R vector DNA and the 30 selection genes conferring resistance to the antibiotics G418, phleomycin or hygromycin B. Briefly, the experimental set up was as follows (Eigure 12):

1) one-step gene disruption of a SIT4 gene via transformation of yeast with pGb-RBN3 digested will SfiI.

~T
~.~

- 31 - l 335264 Transformants have been selected for resistance to G418. The outcome was strain ApGb-RBN3, in which a SIT4 gene has been re-placed by SIT4 gene, interrupted by the G418r gene under control of the ADHI promoter.

2) strain ApGb-RBN3 was used as host strain in a co-transformation protocol with pUT332 and with pGb-RBRR01 digested with SfiI. pUT332 is a 2~-derived episomal plasmid containing a gene conferring resistance to phleomycin. The first selection in this cotransformation step was carried out on plates contain-ing phleomycin (30 ~g/ml). In a certain percentage of these phleomycinr yeast cells (in the order of 0.1 to 1%) the inter-rupted SIT4 gene (with pADHI/G418r) has been replaced by the co-transformed SIT4 fragment containing altered maltose permease 15 and maltase genes. This second gene-replacement event resulted in a yeast which is again sensitive to G418. To select for yeast cells in which this second gene-replacement has taken place, phleomycinr transformants were replica-plated onto plates containing G418 (300 ~g/ml). In cells which did not grow, the 20 G418r gene embedded between SIT4 gene sequences, has been re-placed by the altered MAL-genes, embedded between SIT4 gene se-quences.

3) phleomycinr G418SenS transformants were then 25 cured from the episomal plasmid pUT332 by growth on non-selec-tive medium (i.e. without phleomycin) during 10-20 generations.
The resulting transformant ApGb-pRBRR01 is sensitive to both phleomycin and G418 and contains no prokaryotic sequences. All the integration events have been verified at the DNA level with 30 Southern blot experiments (data now shown).
The resulting strain can be used as host in subse-quent transformations by use of another integration locus and/or - in case of yeast strains which are diploid or polyploid - at the other allele(s) of the SIT4 gene. Strain A iS aneuploid and X

- 32 - l 335264 diploid for the chromosome containing the SIT4 gene. Both SIT4 genes of strain A were used as target site for gene replacement.
This ultimately yielded transformant ApGb-p2RBRR01#1. The use of both alleles for integration most likely also increased the 5 genetic stability of the transformant, since the second integra-tion event removed the polymorphism at that locus. Such poly-morphic regions are sensitive to gene conversions which can re-sult in the loss of the integrated se~uences. The transformant obtained in this way has been analyzed with Southern blot tech-10 niques and shows the hybridization pattern as predicted from thegene disruption events at both SIT4 genes.
The constructed vector pGB-RB2 which served as star-ting plasmid for pGb-RBN3 and pGb-RBRR01, has several useful characteristics inspired by the following considerations:
1. Often a DNA segment has to be integrated, which is constructed by fusing a yeast promoter to a (yeast) coding region of another gene. Gene replacements are of course only readily obtained if the transforming fragments possess on both 20 sides sequences homologous to a target-sequence in the genome.
Therefore, a DNA segment has to be hooked onto the 3~ end of the coding region (vide supra), which is derived from the same gene as the selected promoters. The disadvantage of this approach is that it requires many cloning manipulations, since not always the same promoter is used. In addition, often a strong promoter is selected, derived from a gene, which is functional at the stages of interest (during vegetative growth, anaerobic fermen-tation, etc.). After integration of the engineered fragment one copy of this gene is made non-functional.
Therefore, we wish to direct gene replacement at a locus, which is not expressed during vegetative growth. We have selected the SIT4 gene (sporulation-induced transcribed se-quence), whose expression is well studied by Gottlin-Ninfa - 33 - l 335264 and Kaback (supra), but of course other SIT genes or in general 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, in which the gene is located, is transcriptionally inactive during vege-tative growth as a result of a silencer, analogous to the one described for the HMR-locus (A.H. Brand et al. (1985) Cell 41, 41-48). This silencer DNA also represses transcription con-trolled by promoters, unrelated to mating-type promoters and can act on promoters 2600 bp away. Gottlin-Ninfa and Kaback (sup-ra), however, have shown that the HIS3 gene is able to function during vegetative growth when integrated into the SIT4 gene.

2. To facilitate cloning manipulations, the SIT4 gene is provided with a synthetic piece of DNA, containing sev-eral unique restriction sites (polylinker with cloning sites).
In addition, the polylinker contains at both sides stopcodons for translation in all possible reading frames (see Figure 9).
20 This is a safety-valve to stop translation of any possible hy-brid transcript, which may be synthesized across the junction.
Such a hybrid transcript may otherwise code for a protein, whose effect is unknown.

3. To achieve homologous recombination, the DNA seg-ment to be integrated contains at both ends restriction sites for NotI and SfiI, which both recognize 8 bp sequences. The frequency of occurrence of these restriction sites is very low and hence it is extremely unlikely, that a DNA segment to be cloned into the polylinker in the SIT4 gene, contains both rec-ognition sites. Thus, once the DNA segment has been cloned into the SIT4 gene on plasmid pGb-RB2, restriction with NotI or SfiI
liberates a DNA fragment, which can recombine by interacting with homologous sequences in the genome, i.e. at the SIT4 locus.

_ 34 _ l 3 3 5 2 6 4 Although the very ends are part of the NotI or SfiI site and hence not homologous, other studies have shown that - apparently by limited exonuclease digestion in the cell - these sequences are removed (e.g. H. Rudolph, J. Koenig-Ranseo and A. Hinnen (1985) Gene 36, 87-95).

4. As starting plasmid the commercially available plasmid pTZ19R, a pUCl9 derivative is used. This vector has the advantage that it has a high copy number and that it possesses 10 an origin of replication of the single-stranded phage fl. this makes it very easy to isolate - after infection with a helper phage - single-stranded DNA and to verify the DNA sequences across the junctions with the aid of primers. This greatly fac-ilitates the detailed description of manipulated DNA.
It will be appreciated that it is possible to apply these improvements not only to bakers' yeast, but to other yeasts as well.

20 CO2-~roduction measurements a) in synthetic dough medium (Test A) Yeast cells were incubated in YEPMS medium (1% yeast 25 extract; 2% bactopepton; 3.75% maltose; 1.25% sucrose supple-mented with 200 ~g/ml G418). Growth was at 30C until late-log phase. Yeast cells were collected from 6 ml culture and resus-pended in 8.8 ml synthetic dough medium. Composition of this medium (per litre): saccharose 4.6 g; maltose 64.37 g; KH2PO4 30 2.07 g; MgSO4.7H2O 2.76 g; (NH4)2SO4 0.67 g; casamino acids 2.07 g; citric acid 4.02 g; Na3 citrate 44.25 g; vitamin Bl 9.2 mg;
vitamin B6 9.2 mg; nicotinic acid 46 mg; Ca-D(+)-pantothenate 18.2 mg; biotin 0.23 ~g. During 10 minutes the suspension was allowed to equilibrate in a 28C waterbath, with moderate stir-35 ring after which the flasks containing the yeast suspension wereconnected via a tube to a "gasourette".

~ 35 ~ 1 3352 64 This burette was filled with a solution containing per liter 20 ml indicator solution (1 g methylred; 0.5 g methylenblue;
dissolved in 1 1 96% ethanol), 40 ml lN H2~04 and a trace of CuS04 (dissolved in HN03). The displacement of the volume of 5 this solution in the burette is a measure for the C02-production, which was measured during 165 minutes.
In each set of experiments values of C02 production obtained have been corrected for environmental temperature and pressure to standard conditions of ~8C and 760 mm ~g, 10 respectively. In addition, a correction was made for the amount of yeast. The colorimetric readings at 600 nm of the culture have been used as correction factor in order to equalize the amount of yeast per C02 measurement.

15 b) in dough (Test B and B') m e C02 gasslng curves of compressed yeast grown fed-batch wise on molasses was determined, in dough with no sugar added (lean dough) (Test B) or with 30% sugar (Test B').
The lean dough was prepared as follows: 1 g of compressed 2~ yeast (containing 28.5% dry matter), 34 ml salt solution A
(1.25 g NaCl dissolved in 34 ml of dlstilled water) and 62.5 g flour were mixed in a Hobart apparatus for 30 seconds at speed 1 and 2 minutes at speed 2 so as to obtain a well developed dough.
The ~0% sugar dough contained 2.0 g of compressed yeast (of 28.5% dry matter), 34 ml salt solution B (O.g3~ g NaCl in 34 ml of d~stilled water), 62.5 g flour and 18.75 g sucrose (i.e. 30% sugar with respect to flour). Mixing was as for lean dough.
~le dough was then transferred to a round-bottom flask. ~as-production measurement was started 7.5 minutes after mixing, by connecting the flasks containing the dough via a tube to a gasburette (see section a) and performed during 165 minutes at 28C. In the event that the dry matter 35 content of the compressed yeast differs from the value indicated above, such yeast has been employed; however, the - 36 _ 1335264 measured value of the CO2 gassing power has then be corrected by multiplying the CO2 gassing power by the ratio of the required dry matter content to the actual measured value. In each set of experiments, values of CO2 production obtained have been correc-5 ted for environmental temperature and pressure to 28C and 760mm Hg, respectively. In some cases additional calculations have been performed in which the gas values obtained have been cor-rected for the percentage of N of the fed-batch grown yeast (% N
is an indication of the protein content). Similar percentages 10 of improvements were found as without this last correction.

c) in dough (Test C and C') The CO2 gassing curves of dried yeast, such as in-15 stant dry yeast prepared according to procedures as described inU.S. Patent No. 3,843,800 and U.S. 4,341,871 was determined in dough with no sugar added (test C) or with 30% sugar (test C').
These tests were performed in the same way as described in tests B and B' except that 300 mg of dried yeast (containing 96% dry 20 matter) and 600 mg of dried yeast (containing 96% dry matter) were used in tests c and c~, respectively. Prior to the test the yeast was mixed with the flour and incubated for 10 minutes at 28C.

25 F;~n7vmatic analvses The capacity to transport maltose by yeast cells was determined using [U-14C]-maltose at a concentration of 15 mM as a substrate at 30C. Details have been published by R . Serrano (Supra). Maltase (E.C. 3.2.1.20) was assayed for using p-nitro-phenyl-a-D-glucopyranoside as a substrate in cell free extracts.
The assay was carried out according to H. Halvorson and E.L.
Elias, Biochim. Biophys. Acta (1958) 30, 28.

_ 37 _ l 33 52 6 4 Substrate consumption and product formation in liquid medium The disappearance of maltose and glucose rrom liquid media was quantitated using standard HPLC-techniques.
5 One litre of medium contained: 100 g maltose, 10 g glucose, 3-0 g (NH4)2S04, 4.0 g Mgso4.7H2o~ 4 g KH2P04~ 4 g casamino acids (Difco), 4 g citric acid.H20, 45 g trisodiumcltrate.2H20, 10 mg vitamin Bl, 10 mg vitamin B6, 40 mg nicotinic acid, 20 mg Ca-D(+)-pantothenate and 0.02 mg 10 biotin. The pH was adjusted to 5.7. Two ml of medium was added to a suspension of yeast (20 mg dry weight/2.0 ml of distilled water). This mixture, termed medium A, was incubated at 28C.
Medium B was like medium A but contained 20 times more glucose. The experiment was carried out under anaerobic 15 conditions.

Protein determination Protein of cell-free extracts and of whole cells 20 was determined by the microbiuret method of J. Goa, Scand. J.
Chim. Lab. Invest. (1953) 5, 218. Ovalbumin served as a standard.

Keepin~ quality C~ompressed yeast was stored ln closed plastlc containers at 23C during 4 days. Keeping quality is defined as the percentage of remaining gassing power after this period.
Manufacture of compres~ed yeast A culture Or a yeast strain was grown in a series of fermentors. Cells were cultivated in 10 litre laboratory 35 fermentors with a net volume of 6 litres. During the fermentation pH and temperature were maintained at desired values by automatic control. The fermentation recipe used is based on procedures described by G. Butscheck and R. Kautzmann, Die Hefen, Band II Technologie der Hefen p. 501-591 (1962), Verlag Hans Carl, N~rnberg, FRG and those published by G. Reed 5 and H.J. Peppler in Yeast Technology, the AVI Publishing Company Inc., Westport, Connecticut, U.S.A. (1973). The cultivation conditions of the final fermentation were in particular:
- molasses applied consisted of 80% by weight of beet molasses and 20% by weight of cane molasses, calculated on 10 the basis of 50% sugar.
- the required amount of phosphate was added in the form of mono-ammonium phosphate, prior to inoculation.
- the temperature increased from 28C to 30C during the fermentation according to Table 1.
- nitrogen was supplied during the fermentation as a 10% solution of NH3 in water according to Table 1.
- pH was kept at 5.0 during the first 8 hours of the fermentation and increased thereafter according to Table 1 to 6.2 at the end of the fermentation.
- per kg of molasses containing 50% fermentable sug-ars 12 mg of vitamin Bl was added prior to inoculation.
The yeast obtained by this fermentation was concen-trated and washed with tap water in a laboratory nozzle centri-fuge. Yeast creams were compressed to a dry matter content var-25 -ying between 26 and 32%.
The obtained protein content (% N x 6.25) varied be-tween 42-55% of dry weight as a conse~uence of different quanti-ties of ammonia applied during the fermentation.

- 39 - l 335264 Table 1 Fermentation recipe used for the fed-batch wise production of baker's veast Hours after Molasses supply pH T Ammonia supply inoculation t% of total (C) (% of total amount added) amount added) 0 7 5 28.0 0 0- 1 - 5 28.0 0 1- 2 5 5 28.0 0 2- 3 6 5 28.5 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 C2 production of yeast transformed with 2~-derived plasmids cont~; n i ng genes derived from the MAL6 locus Commercial baker's yeast strains A and C have been transformed with pGb-eMAL6g (maltose permease and maltase, see Fig. 1). pGb-eMAL61 (maltose permease, see Fig. 2) and pGb-eMAL63 (MAL-regulator, see Fig. 3). The MAL genes still contain 10 their original promoters. The nomenclature of the transformed yeast strains is as follows: ApeG418 denotes strain A trans-formed with peG418. Other transformed strains have been indi-cated in an analogous way. The effects of these plasmids on the C2 production in synthetic dough medium is summarized in Table 2. The host strain, transformed with starting peG418 (see Fig.
1) serves as a reference, since we have found that the mere pre-sence of a multicopy plasmid has a negative effect on the gas production.
All transformants display major CO2 production im-20 provements, relative to the control. The combination of extracopies o~ both maltose permease and maltase genes gives the highest enhancement, about 40% in strain A and 18% in strain C.
Transformants ApGb-eMAL6g and CpGb-eMAL6g have also been tested in dough with no added sugar. In this case the 25 yeast was grown fed-batch wise on molasses.
Again, major improvements in CO2 production were ob-tained (Table 3).

.

~ 335264 Table 2 Gas production of strain A and strain C transformed with 2~-de-rived MAL plasmids, relative to the vector-transformed strains.
5 C2 production was measured in synthetic dough medium (see ex-perimental procedures) and corrected to 285 mg dry matter. Data are mean values of several experiments.

10 strains 100 minutes 165 minutes ApeG418 100 100 ApGb-eMAL6g 157 141 ApGb-eMAL61 144 123 15 ApGb-eMAL63 119 115 CpeG418 100 100 CpGb-eMAL6g 121 118 CpGb-eMAL61 117 114 Table 3 Relative gas production in dough of strains A and C, transformed 25 with 2~-derived plasmids peG418 and pGb-eMAL6g, C02 production was corrected to 285 mg dry matter.

strains 60 100 120165 minutes minutes minutes minutes ApeG418 100 100 100100 ApGb-eMAL6g125 125 125121 35 CpeG418 100 100 100100 CpGb-eMAL6g151 141 138129 - 42 - ~ 3 3 52 6 4 Example 2 C2 production of yeast strains transformed with integrating plasmids containing recombinant maltase and/or maltose permease 5 genes Parental yeast strain A has been transformed with pGb-iA32/G418 (main feature: ADHI/maltose permease; see Figure 6) and pGb-iRR01 (main feature: ADHI/maltose permease and 10 EFl~A/maltase; see Figure 7).
Parental strain A and both integrative transformants are grown fed-batch wise on molasses similar to the commercial aerobic fermentation (see Table 1). Ater harvesting the cells, C2 production is measured in a standard dough test with no sug-15 ar added. The gas production, as analyzed in this test, is sum-marized in Table 4. Integration of pGb-iA32/G418 into the chro-mosome of strain A improves gas production in dough significant-ly. The relative improvement varies somewhat depending on time of measurement (see Table 4). When in addition to an altered 20 maltose permease gene an altered maltase gene is integrated in the chromosome of commercial strain A using pGb-iRR01 gassing power is even further improved. In this typical experiment ab-out 30% more CO2 is produced after 165 minutes in a lean dough, which corresponds to a level of about 410 ml C02/285 mg dry 25 weight of yeast.
In a 30% dosage sugar dough no substantially differ-ences in CO2 production of the transformants were noticed com-pared to the parental strain (about 190 ml C02/285 mg dry weight of yeast).
The obtained improvement in leavening activity is maintained during storage at 23C. The loss of leavening ac-tivity during storage is virtually identical for parental strain A and the novel strains (see Table 5).
X

- 43 - l 3 3 5264 Table 4 Relative gas production of strain A and its rDNA derivatives provided with altered maltase and/or maltose permease genes.
5 Gas values have been corrected to 285 mg dry matter. No sugar was added to the dough.

Strain 60 100 120 165 minutes minutes minutes minutes ApGb-iA32/G418 113 115 115 111 ApGb-iRR01 131 136 138 133 Table 5 Keeping quality of strain A and its rDNA derivatives provided 20 with altered maltase and/or maltose permease genes. Leavening activity was measured as in Table 4 after keeping of compressed yeast at 23C ~or 4 days 25 Strain keeping quality (% of original leavening activity) ApGb-iA32/G418 91 30 ApGb-iRR01 88 - 44 - l 335264 Example 3 C2 production of a yeast strain which contains recombinant mal- =
tase and maltose permease genes and no heterologous DNA.
Parental strain A has been genetically modified such that a pADHI/maltose permease gene and a pEFl~A/maltase gene were introduced into the SIT4 gene on both homologous chromo-somes. This strain, abbreviated ApGb-p2RBRR01 #1, has been con-structed using the methods and plasmids as described previously(see sections of transformation and construction of plasmid of pGb-RBN3 (Fig. 10) and pGb-RBRR01 (Fig. 11) and the general scheme of transformation via gene-replacement. Parental strain A and the homologous transformant ApGb-p2RBRR01 #1 have been 15 grown fed-batch wise on molasses similar to the commercial aero-bic fermentation (see Table 1). After harvesting the cells, CO2 production is measured in a standard dough test with no sugar added.
Table 6 summarizes the results. In the ~lean~ dough 20 the improvement is about 18% after 165 minutes, which corres-ponds to a level o~ about 367 ml C02/285 mg dry weight of yeast.
This strain contains two copies each o a maltose permease gene under control of the ADHI promoter and a maltase gene under con-trol of the EFl~A promoter. In Table 4 is shown that strain 25 ApGb-iRR01 has an improvement of about 30%. An initial estima-tion of the number of pGb-iRR01 molecules integrated into a MAL-locus of 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-30 p2RBRR01 #1 (e.g. by integration in other sporulation-specific genes) a homologous transformed yeast strain can be obtained with at least similar gas production levels as strain ApGb-iRR01.

_ 45 _ ~ 33526 4 Table 6 Relative gas production of strain A and its homologous rDNA
derivative provided with altered maltase and maltose permease 5 genes. Gas values have been corrected to 285 mg dry matter. No sugar was added to the dough.

Strain 60 100 120 165 minutes minutes minutes minutes ApGb-p2RsRR01 #1 124 128 127 118 . ..

Example 4 Enzyme activities and substrate uptake rates of yeast strains transformed with integrating plasmids containing recombinant 5 maltase and/or maltose permease genes As described above, the expression of maltose perm-ease and maltase is subject to maltose induction and glucose 10 repression. This phenomenon is shown in Fig. 13 for wild-type cells of strain A. Specific activities of maltose permease and maltase do not increase until most of the glucose has been util-ized.
The activity of maltose permease at the onset of 15 dough-rise is increased by introduction of an altered maltose permease gene into the yeast genome (strain ApGb-iA32/G418) as shown in Fig. 14.
Surprisingly, activities of maltase were increased as well in this construct (Fig. 15). This novel strain ApGb-20 iA32/G418 fermented maltose more rapidly than the parental strain A in medium A which contains maltose as main carbon and energy source (Fig. 16). In medium B containing glucose as main carbon and energy source this effect was less pronounced (Fig.
17).
In addition to an altered maltose permease gene an altered maltase gene was integrated in the chromosome yielding strain ApGb-iRR01. This strain fermented maltose at an even higher rate in medium A (Fig. 16) and also in medium s (Fig.
17). Despite the high extra extracellular concentration of glu-30 cose considerable amounts of maltose were metabolized by this novel strain. Strain ApGb-iRR01 exhibited higher specific acti-vities of maltase and maltose permease during dough-rise than parental strain A and strain ApGb-iA32/G418 (Fig. 14 and 15).

Claims (68)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as fol-lows:

1. A transformed yeast capable of enhanced produc-tion of carbon dioxide as compared to an untransformed par-ent of said yeast upon fermentation in a medium comprising maltose, wherein said maltose is fermentable by both said parent and said transformed yeast, and wherein said trans-formed yeast comprises a DNA construct comprising at least one gene capable of expression in said transformed yeast encoding a maltose permease, maltase or a maltose regula-tory protein.

2. A yeast according to claim 1 wherein said con-struct comprises at least one gene which encodes an enzyme having maltose permease activity, maltase activity or mal-tose regulatory protein activity, or any combination there-of.

3. A yeast according to claim 2 wherein said genes or combinations thereof have been brought under tran-scriptional control which is not sensitive to glucose re-pression and is not subject to maltose induction.

4. A yeast according to claim 2 wherein said gene is under transcriptional control of alcohol dehydrogenase I
(ADHI) and/or translation elongation factor (EF1.alpha.A) promo-ter.

5. A yeast according to claim 4 wherein said pro-moters are derived from a yeast belonging to the genus Sac-charomyces.

6. A yeast according to claim 5 wherein said Sac-charomyces is Saccharomyces cerevisiae.

7. A yeast according to claim 1, 2, 3, 4, 5 or 6 wherein said construct is a portion of an episomal element.

8. A yeast according to claim 7 wherein said con-struct is integrated into a chromosome of said yeast.

9. A yeast according to claim 8 which comprises at least two of said constructs.

10. A yeast according to claim 9 wherein said yeast is free of heterologous DNA.

11. A yeast according to claim 1 wherein said con-struct comprises at least two said genes.

12. A yeast according to claim 1 wherein transcrip-tion of said gene is regulated by a transcriptional initia-tion region foreign to said gene.

13. A yeast according to claim 1 wherein said gene is under the transcriptional initiation control of the yeast alcohol dehydrogenase I promoter or yeast translation elongation factor promoter.

14. A yeast according to claim 1 wherein said con-struct is a portion of a plasmid stable in said yeast.

15. A yeast according to claim 1 wherein said con-struct is integrated into a chromosome of said yeast.

16. A yeast according to claim 1 wherein said con-struct is free of prokaryotic DNA.

17. A yeast according to claim 1 wherein said yeast is of the species Saccharomyces cerevisiae.

18. A transformed yeast capable of enhanced fermen-tation of maltose into ethanol and carbon dioxide as com-pared to an untransformed parent yeast capable of fermen-ting maltose wherein said transformed yeast comprises a DNA
construct comprising at least one of a gene encoding mal-tase, maltose permease or a maltose regulatory protein, wherein transcription of at least one of said genes is reg-ulated by a constitutive promoter functional in yeast and wherein said DNA construct is free of prokaryotic DNA.

19. A yeast according to claim 18 wherein maltase is brought under transcriptional gene expression control of a translation elongation factor (EF1.alpha.A) promoter.

20. A yeast according to claim 18 wherein maltose permease is brought under transcriptional gene expression control of an alcohol dehydrogenase I (ADHI) promoter.

21. A yeast according to claim 18, 19 or 20 where-in said promoters are derived from a yeast belonging to the genus Saccharomyces.

22. A yeast having a moisture content of 3 to 8%
which is produced by drying a yeast obtained according to claim 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

23. A yeast according to claim 22 wherein said yeast is belonging to the genus Saccharomyces.

24. A yeast according to claim 23 wherein said Sac-charomyces is Saccharomyces cerevisiae.

25. A yeast obtainable by strain improvement pro-cedures other than DNA mediated transformation using a yeast according to claim 23.

26. A yeast selected from the group consisting of 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 CpGb-eMAL6g, Saccharomyces cerevisiae strain CpGb-eMAL61, and Saccharomyces cerevisiae strain ApGb-p2RBRR01#1.

27. A compressed, instant dry or active dry yeast obtainable from a yeast according to claim 23, 24, 25 or 26.

28. A yeast according to claim 18 wherein said pro-moter is a yeast promoter and comprises an alcohol dehydro-genase I promoter or a translation elongation factor-1 or -2.

29. A yeast according to claim 27 wherein said yeast promoter is a Saccharomyces promoter.

30. A yeast according to claim 1, 11, 12, 13, 14, 15, 16, 17, 18, 28 or 29 having a moisture content of 4 to 8%.

31. The transformed yeast according to claim 1 wherein said gene is homologous to said host.

32. The transformed yeast according to claim 1 wherein said gene is a chimeric gene.

33. The transformed yeast according to claim 32 wherein the promoter of said chimeric gene is a constitu-tive promoter.

34. A yeast according to claim 1 wherein said med-ium is flour or dough.

35. A transformed yeast capable of enhanced produc-tion of carbon dioxide as compared to an untransformed par-ent upon fermentation under anaerobic conditions in a med-ium comprising maltose, wherein said maltose is fermentable by both said parent and said transformed yeast, and wherein said transformed yeast comprises a DNA construct comprising at least one gene capable of expression in said transformed yeast encoding a maltose permease, maltase or a maltose reg-ulatory protein.

36. A yeast according to claim 35 wherein said fer-mentation under anaerobic conditions comprises fermentation in dough.

37. A Saccharomyces cerevisiae strain selected from the group consisting of ApGb-iA32/G418, ApGb-iRRol, ApGb-eMAL6g, ApGb-eMAL61, ApGb-eMAL63, and CpGb-eMAL6g.

38. A plasmid selected from the group consisting of pGB-iA32/G418, pGB-iRRol, pGB-eMAL6g, pGB-eMAL61, pGB-eMAL63, and pGB-M6g(delta-9).

39. A compressed yeast, obtainable from a trans-formed yeast according to claim 1, which shows a gas pro-duction of at least 340 ml/285 mg dry weight of yeast in 165 minutes in Test B and a gas production of at least 170 ml/285 mg dry weight of yeast in 165 minutes in Test B'.

40. A compressed yeast, obtainable from a trans-formed yeast according to claim 1, which shows a gas pro-duction of 380-450 ml/285 mg dry weight of yeast in Test B
in 165 minutes and a gas production of 180-240 ml/285 mg dry weight of yeast in 165 minutes in Test B'.

41. A compressed yeast, obtainable from a trans-formed yeast according to claim 1 which shows a gas pro-duction of at least 400 ml/285 mg dry weight of yeast in 165 minutes in Test B and a gas production of at least 190 ml/285 mg dry weight of yeast in 165 minutes in Test B'.

42. A compressed yeast, obtainable from a trans-formed yeast according to claim 1, which shows a gas pro-duction of 400-500 ml/285 mg dry wight of yeast in 165 minutes in Test B.

43. A compressed yeast, obtainable from a trans-formed yeast according to claim 1, which shows a gas pro-duction of 440 ml/285 mg dry weight of yeast in 165 minutes in Test B.

44. An instant dry or an active dry yeast obtain-able by drying the compressed yeast according to claim 39, 40, 41, 42 or 43.

45. A dried yeast, obtainable from a transformed yeast according to claim 1, which shows a gas production of 310-360 ml/285 mg dry weight of yeast in 165 minutes in Test C and a gas production of 145-195 ml/285 mg dry weight of yeast in 165 minutes in Test C'.

46. A dried yeast, obtainable from a transformed yeast according to claim 1, which shows a gas production of at least 330 ml/285 mg dry weight of yeast in 165 minutes in Test C and a gas production of at least 155 ml/285 mg dry weight of yeast in 165 minutes in Test C'.

47. A dried yeast, obtainable from a transformed yeast according to claim 1, which shows a/gas production of 320-400 ml/285 mg dry weight of yeast in 165 minutes in Test C.

48. A dried yeast, obtainable from a transformed yeast according to claim 1, which shows a gas production of at least 350 ml/285 mg dry weight of yeast in 165 minutes in Test C.

49. A vector selected from the group consisting of pGb-eMAL6g, pGb-eMAL61, pGb-eMAL63, pGb-iA32/G418, pGb-iRRol, pGb-M6g(delta-9), pGb-RBRR01, pGb-RBN3, and pGb-RBREG01.

50. A dough or a similar product which comprises a yeast according to claim 23, 24, 25, 26, 39, 40, 41, 42, 43, 45, 46, 47, 48 or 49.

51. A process to produce a leavened flour product, or an alcoholic beverage or other alcoholic product which comprises the use of a yeast according to claim 23, 24, 25, 26, 39, 40, 41, 42, 43, 45, 46, 47, 48 or 49.

52. A process to produce a transformed yeast cap-able of enhanced production of carbon dioxide as compared to an untransformed parent of said yeast upon fermentation in a medium comprising maltose, wherein said maltose is fer-mentable by both said parent and said transformed yeast which comprises the introduction into a yeast of at least one homologous DNA construct which comprises at least one gene encoding a protein promoting the uptake and/or initial metabolic conversion of a transported substrate, said intro-duction comprising DNA mediated transformation or other me-thod of strain improvement.

53. A process according to claim 52 wherein the construct comprises at least one gene which encodes an en-zyme having maltase activity, maltose permease activity or maltose regulatory protein activity.

54. A process according to claim 53 wherein the construct comprises at least two of said genes.

55. A process according to claim 53 wherein said gene is under transcriptional control of alcohol dehydro-genase I (ADHI) and/or translation elongation factor (EF1.alpha.A) promoters, respectively.

56. A process according to claim 52, 53, 54 or 55 wherein said gene or combinations thereof have been brought under transcriptional control which is not sensitive to glucose repression and/or is not subject to maltose induc-tion.

57. A process according to claim 56 wherein said construct is a portion of an episomal element.

58. A process according to claim 57 wherein said construct is integrated into a chromosome of said yeast.

59. A process according to claim 58 which com-prises the introduction of at least two of said DNA con-structs.

60. A process according to claim 52, 53, 54, 55, 57, 58 or 59 wherein said yeast is free of heterologous DNA
which comprises the integration of genes into the chromo-some using gene replacement techniques.

61. A process according to claim 60 wherein a chro-mosomal sporulation-specific gene is replaced by a DNA seg-ment comprising the same identical sporulation-specific gene into which genes described in claim 52, 53, 54, 55, 57, 58 or 59 have been inserted.

62. A process according to claim 55 wherein said promoters are derived from a yeast belonging-to the genus Saccharomyces.

63. A process according to claim 52, 53, 54, 55, 57, 58, 59 or 62 wherein said yeast belongs to the genus Saccharomyces.

64. A process according to claim 63 wherein said Saccharomyces is Saccharomyces cerevisiae.

65. A process to produce a dough or similar pro-ducts which comprises the application of yeast produced according to claim 63.

66. A process to produce alcoholic beverages and other alcoholic products which comprises the use of yeast produced according to claim 63.

67. A process to produce bread and related pro-ducts which comprises the use of yeast produced according to claim 63.

68. A process to produce an enzyme with maltase or maltose permease activity which comprises the use of yeast produced according to claim 63.