AU612112B2 - Block for the expression of an aminolucosidase in yeast, transformed yeast, enzyme preparation process and fermentation process - Google Patents

Description

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612 412~ P/00/011 PATENTS ACT 1952-1973 COMPLET,,E PECIFICATION

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FOR OFFICE USE Form Class: Int. Cl: Application Number: Lodged: Complete Specification-Lodged: 9*

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Accepted: Published: 4 r 4 Priority: Related Art: Name of Applicant: TO BE COMPLETED BY APPLICANT TRANSGENE a French body corporate, of 16 rue Henri Regnault, 92400 Courbevoie, France.

Address of Applicant: Actual Inventor: NATHALIE LABAT GERARD LOISON MARTINE NGUYEN-JUILLERET YVES LEMOINE Address for Service: I1 QUVENS RO0AD Mg~l OURNE, 5004, AUSI RA" Complete Specification for the invention entitled: AA BLOCK FOR THEzEXPRESSION OF A A1IOLC3DC IN A YEAST, TRANSFORMED YEAST, ENZYME PRF.PARATIO LPROCES AND FERMNTAON PR CESS Thefolowig sateentisa fll escipton his invention, including the best met iod of performing it knownt tome:' 'Note: The description is to be typed in doubia type facerna ae o le spaci', pica ,i nae o exceeding 250 mm in dsept and IGO inm ini widtht, on tough white paper oii good quality'and it is to be inserted inside thjis form.

117 ;0/76-L C.J, 4 The present invention describes processes by means of which different strains of yeast of the genus Saccharomyces, q including the industrial strains which do not have the capacity to degrade complex carbon-containing substrates such as starch or dextrins, may be caused to secrete a glucoamylase.

Starch, which is commonl) used as a source of carbon in alcoholic fermentation, has to be hydrolyzed before- Shand (either chemically or enzymatically) in a separate process in order to produce substrates which are usable for fermentative yeasts.

It would hence be desirable to construct, by genetic recombination, industrial strains which were capable of synthesizing one or more enzymes for degrading starch to substrates which are directly assimilable by these strains.

More especially, the objective of the present invention is to construct, by genetic recombination, a strain responsible for brewery fermentation capable of degrading the S residual dextrins in the wort so as to produce beers having a low content of sugars.

cc '0 Similarly, the enrichment of the wort with respect

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to fermentable sugars also enables the production of beers having a high alcohol content to be envisaged.

The use of a brewery strain which produces an enzyme responsible for the degradation of dextrins during alcoholic fermentation may prove to be a means of simplifying the brewing processes and making them profitable.

The preparation of beers having a low content of residual sugars can, in particular, be achieved by adding, at various stages of the brewing process, an Aspergillus niger glucoamylase (amylo-1,6-glucosidase). This glycoamylase catalyses the release of glucose residues from the non-reducing ends of starch ,r dextrins.

This enzyme plays a big part in the production of S glucose syrups from starch and in the manufacture of other alcoholic drinks. This Aspergillus niger glucoamylase is i; accepted as an additive in the agri-foodstuffs industries.

Hence, in the first place, the subject of the present invention is the expression of Aspergillu.s niger NC122343 4 *4 -2 1 1 i r 1 1 1 alpha-amyloglucosidase in a brewery strain.

Secondly, one of the advantageous features of the invention is that the transformed brewery strains possess the foreign DNA integrated in a yeast chromosome. When the foreign DNA is integrated in the chromosome according to the process described Later, the property of producing alpha-amyloglucosidase is stable and the strain differs from the starting strain only in respect of this new character.

The stability of the information can also be achieved by constructing a DNA structure known as a minichromosome consisting of three main elements: telomere, centromere and origin of replication.

Another subject of the present invention relates, more especially, to a functional DNA block enabling amyloglucosidase to be secreted from yeast, this block containing the following features: 1 e S. the amyloglucosidase gene devoid of all or part of its introns; a DNA sequence containing the signals which provide for the 20 transcription of the amyloglucosidase gene by yeast; a coding sequence containing the signal required for the export of the mature eavloglucosidase into the extracellular medium; the choice was made of a secretion system specific to yeast, that of the alpha pheromone but other systems could be used Efor example, the killer protein system or that of a synthetic signal sequence]; finally, the expression block may possess, after the amyloglucosidase gene, a yeast termination sequence, for example that of the phosphoglycerate kinase gene.

30 Preferably, the amyloglucosidase gene will be dei void of all of its introns (cDNA).

Among the elements of the sequence which provide for the transcription of the amyloglucosidase gene, promoters should be mentioned, in particular the PGK promoter.

This functional block can be integrated in an autonomously replicating plasmid containing, for example, an origin of replication in yeast, for example Lthe origin of replication of the 2 pla'mid.

0 i 3 When such a plasmid is used, it is advantageous that it should be a shuttle plasmid capable of replicating in a bacterium; for this purpose, it may contain elements for rep- Lication in a bacterium, for example E. coli, and a selection gene such as that for resistance to an antibiotic, for example Amp r In general, the plasmid will preferably contain an effective gene for selection in yeasts, for example a gene complementing an auxotrophy, such as ura 3 or alternatively a gene conferring a dominant phenotype, for example resistance to G418.

When the expression block is integrated in the genome of the yeast strain, the integration will be performed in a site which is nonessential for the yeast, for example the MFalphaI gene.

The constitution of an integration vector is known and will be described in detail in the examples.

t The invention also relates to the strains containing an expression block according to the invention, involving either a plasmid or a chromosomal integration.

«Among the transformed strains, there should be mentioned, in particular, the strains of Saccharomyces, in particular S. cerevisiae and S. uvarum, in particular the indus- S trial strains such as the brewery strains.

Finally, the invention relates to a process for preparing amyloglucosidase, in particular alpha-amyloglucosidase, a wherein a strain according to the invention is fermented on a suitable culture medium and the enzyme is recovered in the extracellular medium.

.'30 The invention relates, finally, to a process for degrading starch or dextrins, wherein an industrial medi containing starch or dextrins is fermented.

Finally, the alpha-amyloglucosidase gene used is preferably that of Aspergillus niger.

Lastly, the combined action of amyloglucosidase and a liquefying alpha-amyLase Leads to a totaL synergy of degra- Sdation of starch to gucose residues. SStarting with industrial strains such as Saccharomyces, ':1 j.'i 4 in whih the genetic information for an alpha-amyloglucosidase and also that for an endo-alpha-amylase (for example, of Bacillus licheniformis) are integrated together in the yeast chromosomes, the production of ethanoL may be obtained using starch as a source of carbon.

Thus, the present invention relates to the introduction of the gene for B. Licheniformis alpha-amylase and its expression in a strain of Saccharomy es producing alpha-amylogLucosidase.

The simultaneous expression of amylogLucosidase and ofalpha-amylase should lead to a synergy of these two enzymatic activities in the degradation of starch or dextrins to glucose residues.

The present invention relates, more especially, to the simultaneous expression of alpha-amylase and amylogluco- Ssidase in a polyploid strain of brewer's yeast, S. uvarum, in particular the strain 2285 TG2 AMGC, as well as to the use of these strains for preparing alpha-amylase.

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660 Moreover, a most especially preferred application 0. 20 according to the invention relates to the transformation of t t cc r particular strains of Saccharomyces, in particular the polyploid and prototrophic strains used in the bread-making industry; such a strain may be, for example, S. cerevisiae strain TGF1.

These polyploid and prototrophic strains have to be cotransformed with a vector as described above and a plasmid which confers a dominant phenotype.

One of the subjects of the present invention is, in effect, the use of alpha-amyloglucosidase, in particular of *30 Aspergillus niger, during panary fermentation, since alphaamyloglucosidase, which degrades starch directly to glucose, has a beneficial effect on panary fermentation since this sugar is directly assimilable by baker's yeast.

The addition of this enzyme enables the baking proi 35 cesses to be improved by making the dough drier to the touch and hence easier to process by machine, and above all enables i the fermentative activity of the baker's yeast to be signifi- I! cantly enhanced and hence the fementation 'times to be +reduced greatly.

Furthermore, this addition should be the source of innovation by enabling relativeLy sweet bread-type products to be developed, which is virtually impossible without adding &n enzyme in a short baking process.

The present invention hence relates, in addition, to processes by means of which a bakery strain of the Saccharomyces cerevisiae type is caused to secrete an amyloglucosidase.

In particular, the invention relates to a panary fermentation process in which at least one of the strains used in the fermentation is a strain as described above.

The examples below are designed to illustrate other characteristics and advantages of the present invention and will be described with reference to the figures, wherein: Figure 1 shows: a) the structure and the restriction map of the Aspergillus niger glucoamylase gene, b) the structure of plasmid pTG1831, c) the structure of plasmid pTG1830, 20 Figure 2 shows the sequence of the Aspergillus niger amyloglucosidase gene, 0 t *0@ 000 fItsc *0I

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0S *025 cc 14 *4444 Figure Figure Figure Figure Figure Figure a) the b) the Figure Figure Figure 3 shows the structure 4 shows the structure and construction of M13TG1843, 5 shows the structure and construction of M13TG1837, 6 shows the structure and construction of M13TG1846, 7 shows the structure and construction of M13TG1848, 8 shows: structure of plasmid pTG1848, structure and construction of plasmid pTG1850, 9 shows the structure and construction of M13TG1857, 10 shows the structure of plasmid pTG1858, 11 shows the detection of the production of amyloof M13TG1839, glucosidase by the strain TGY2sp13 pTG1858 by means of the

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2 /KI test, Figure 12 shows the structure and con struction of plas,id pTG1867, Figure 13 shows the hybridization of the EcoRI restriction fragments of the genomic DNA of the strains TGY2spl3b AMGB -t -6 and TGY2spl3b with the oligonucLeotide TG529 correFsponding to the 3' portion of the MFaLpha 1 gene of S. cerevisiae, Figure 14 shows the hybridization of the restriction fragments of the genomic DNA of the strains TGY2spl3b and 2285TG2 with either the oltigonucLeotide TG529 or the oL igonucLeotide TG170, Figure 15 shows the hybridization of the EcoRI-digested genomic DNAs of strains TGF1-AMG1 and TGF1 with an oligonucleotide TG529 specific for the MFaLphal gene of Saccharomyces cerevisiae, Figure 16 shows the hybridization of the BamHI-digested genomic DNAs of strains 2285 TG2 AMGC-AMY1 and 2285 TG2 AMGC with an oligonucleotide TG227 specific for the His3 gene of S. uvarum.

EXAMPLE 1 CLONING OF THE ASPERGILLUS NIGER ALPHA-AMYLOGLUCOSIDASE GENE

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IN PLASMID pBR322 1 jig of AspergiLLus niger NC122343 genomic DNA and jig of plasmid pBR322 DNA were completely digested with EcoRI and Sal, and these DNAs were then covalently linked using T 4 DNA Ligase. Escherichia coli strain 1106 was then transformed with the ligation products. The selection of the clones carrying an insert corresponding to the alpha-amyoglucosidase gene was carried out after transferring the recombinant clones to a nitrocellulose sheet according to the classical method The recombinant clones were hybridized with a mixture of three oligonuceotides TG282, TG283, TG284, defined on the basis of the sequences already published TG282 5' ATAGTTAAAGGATGGGGATGAGGGC 3' TG283 5' AGGACACGTACTACAACGGCAACCC 3' TG284 5' CTGGGTGACTGGGAAACCAGCGACG 3' Two positive clones were isoated and contain a different EcoRI-SalI fragment. One of the inserts (2.4 kb), which hybridizes with two of the three oligonucLeotides (TG283, -TG284), corresponds to the 5' porrtion of the amytogLucosidase gene. In contrast, the other insert (1 kb), which hybridizes with onLy one of the three otigonucLeotides (TG282), corresponds to <the 3' region of the Aspergitus niger

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f V 7 7 amyloglucosidase gene (Figure 2).

These new pLasmids are referred to as pTG1830 and pTG1831 (Figure 1).

EXAMPLE 2 CLONING OF THE cDNA OF ASPERGILLUS NIGER ALPHA-AMYLOGLUCO- SIDASE INTO BACTERIOPHAGE LAMBDA The sequence of the amyLoglucosidase gene, as weLL as that of the cDNA of AspergiLLus niger (BU-1) amyLog'uwtosidase, has already been pubLished (4a, Comparison of these 'two sequences reveaLs the presence of 4 introi regions in the AspergiLLus niger (BU-1) amyLogLucosidase G1 gene (Figure 2).

With the exception of the intron D, which has a sequence comparable to the consensus sequence TATAAC, an essential eLement in the mechanism of excision of introns in yeast it appears that none of the other introns can be excised by yeast in vivo. CLoning of the cDNA of amyLogLucosidase Et C was hence undertaken.

Li, CL Aspergillus niger NC122343 messenger RNAs were purified from a cuLture of this fungus on rich medium containing '(1120 2% of starch as a carbon source, so as to produce a cDNA corresponding to amyloglucosidase by reverse transcription.

The extracted mRNAs were added to a reaction mixture containing a dT oligonucleotide as a transcription primer.

s The synthesis of the cDNA was carried out according to the C t 4, method described by Chang et at. A cDNA Library was prepared in bacteriophage Lambda according to Le Bouc et at.

2 The bacteriophage Lambda DNA was packaged in vitro in the phage particLes, and the phages thereby formed were used to transduce the Escherichia coli indicator strain POP101.

c c30 The Aspergillus niger NCI22343 cONA library was cccrtte screened for amyloglucosidase, using as a probe an oligonuc- Leotide TG433 defined on the basis of the published sequences (4a): TG433 5'GGTGTAGCCATTGTCAAGCAGCCACTGCCC 3g corresponding to the sequence compLementary to the sequence coding for amino acids 163 to 172 of the mature protein (Figure 2).

Five positive signals were detected. Two of the five Ai i- c;:a 8 clones were confirmed by cross-hybridization with another oligonucleotide TG387 corresponding to the sequence complementary to the sequence coding for amino acids 226 to 236 of the mature protein (Figure 2): TG387 5'CAGGACGAGCCGACGGCCGTCGCGA 3' the size of the inserts -in these two candidates being approximately 800 bp.

After analysis with restriction enzymes, one of the two fragments was recloned in an M13 derivative (M13mp8) in order to sequence it. This new vector is referred to as M13TG1839 (Figure The sequence of the cDNA fragment of M13TG1839 coding for amino acids 62 to 319 of the mature protein proved to be identical to that already published (Figure c I~ 0. r 4 I 4

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EXAMPLE 3 CONSTRUCTION OF AN ALPHA-AMYLOGLUCOSIDASE EXPRESSION VECTOR FOR YEAST S. CEREVISIAE 1) Reconstitution of the 3' portion of the Aspergillus niger arnyloglucosidase gene Plasmid pTG1830 DNA was digested with PstI and Sail so as to liberate a 1.2-kb fragment. Similarly, plasmid pTG1831 DNA was digested with EcoRI and SalI, liberating a 1-kb fragment. These two fragments were isolated and ligated with M13mpS DNA which has been digested with PstI and EcoRI.

A new vector, referred to as M13TG1843, is reconstituted (Figure This vector M13TG1843 possesses the 3' part of the amyloglucosidase gene.

2) Reconstitution of the 5' portion of the region coding for the mature amyloglucosidase of Aspergillus Niger

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1 0i 0c LI a) Removal of the intron A by in vitro mutagenesis In the first place, the BamHI-EcoRI fragment (2 kb) of pTG1830 containing the 5' portion of the amyloglucosidase gene was transferred into M13Tg127 phage DNA between the BamHI and EcoRI sites. This gave single-stranded M13TG1835 DNA, from which the in vitro mutagenesis was carried out. An oligonucleotide TG403 (30-mer), the sequence of which is complementary to those of the regions flanking the intron A, was used as a primer for the synthesis of the DNA, using the single 9 -L i i i -C

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S- 9 strand of M13TG1835 DNA as a template (Figure The sequence of the oligonucleotide TG403 is as follows: TG403 5' ACGGATAACCCGGACTACTTCTACACCT 3' One picomole of single-stranded M13TG1835 DNA was hybridized with one picomole of oligonucleotide TG403 whose ends are phosphorylated in a reaction mixture which contains 10 mM Tris pH 7.5, 10 mM MgCL 2 50 mM NaCl. The mixture was heated to 1000C for 3 minutes and incubated for 1 hour at room temperature.

1.25 il of each deoxynucLeotide triphosphate at a concentration of 5 mM was added to the hybridization mixture in order to obtain a final concentration of 500 uM. 5 units of DNA polymerase I Klenow fragment were then added to one unit of T 4 DNA Ligase; the elongation reaction was continued at room temperature for two hours. After precipitation with ethanol, the Ligation products obtained were digested with the oi restriction enzyme SacI. This enables the frequency of production of recombinant clones containing the mutated form to o, be increased. Competent cells of E. coli strain JM103 were 20 transformed with the digestion mixture. The plaques derived S't from this transformation were analyzed by DNA/DNA hybridization, using as a probe the oligonucleotide TG403 depicted above, labelled with 32P by kinase treatment according to the customary techniques Several positive signals were ob- 25 served. The DNA sequence of one of the clones is identical to that of M13TG1833 DNA without the intron A sequence. This s new phage is referred to as M13TG1837 (Figure b) Construction of the vector M13TG1846 The vector M13TG1837 DNA was digested with BssHII and '0 A 30 Avail. The 380-bp BssHII-AvaII fragment containing the DNA sequence coding for the NH 2 -terminal region of the mature amyloglucosidase of Aspergillus niger (amino acids 21 to 151) was isolated (Figure Similarly, the 280-bp PstI-AvaII fragment of M13TG1839 was isolated. This fragment contains the cDNA sequence coding for amino acids 151 to 256. These two BssHII-AvaII, AvaII-PstI fragments, with two synthetic j oLigonucleotides TG453, TG454 designed to reconstitute the end of the mature amyloglucosidase and which permit the in-phase reading of the prepro sequence of the alpha pheromone followed by the mature sequence of amyloglucosidase (Figure are Ligated in M13mp8 which has been digested with HindIII and Pstl. This new vector is referred to as M13TG1846 (Figure 6).

3) Construction of M13TG1848 In the first place, the HindIII-PstI fragment of M13TG1846 (670 bp) containing the sequence coding for amino acids 21 to 246 was isolated. M13TG1843 DNA was digested with PstI and EcoRV and the 1736-kb PstI-EcoRV fragment derived from this digestion was isolated and eluted from a gel. This fragment contains the 3' portion of the amyloglucosidase gene (presence of the intron D) coding for amino acids 246-640. The EcoRV site is situated downstream from the TGA stop codon (approximately 140 bp). The polyadenyl- S ation site is situated at approximately 70 bp upstream from the EcoRV site. These two fragments are ligated in M13TG131 S41 ttt which has been digested with HindIII and Hindu. This new vector is referred to as M13TG1848 (Figure This new vector was used as a source of amyloglucosidase coding sequence for the secretion vector described below, despite the presence of the last intron D.

4) Construction of an amyloglucosidase expression vector :for yeast S. cerevisiae The starting plasmid is pTG848 (identical to pTG849 described in French Patent Application No. 83/15,716, with the exception of the ura3 gene whose orientation is inverse); it consists of the following fragments (Figure 8a): a) The approximately 3.3-kb EcoRI-HindIII fragment originating from plasmid pJDB207 The HindIII site corresponds to coordinate 105 of the 2 u plasmid B form, the EcoRI site to coordinate 2243. In this fragment, the 2 j is in the B form This fragment enables the plasmid to replicate autonomously in yeast.

b) A HindIII fragment carrying the URA3 gene (11) which enables the plasmid to be selected in ura3- strains on medium jI devoid of a pyrimidine source.

c) The large EcoRI (coordinate 0)-SaI (coordinate 650)

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-11fragment of pBR322 In the PvuII site of this fragment, there has been inserted the 510-bp EcoRI-HindIII fragment corresponding to the end of the PGK gene o(13), the ends of which fragment have been previously rendered blunt by the action of KLenow in the presence of the 4 deoxyribonucleotides. When joined to the PvullI end of pBR322, the flush EcoRI end of the PGK gene regenerates an EcoRI site.

d) The HindIIl -Sall fragment (2.15 kb) of the PGK gene (13).

The BgLII-HindIII fragment of M13TG1848 was isolated and eLuted from gel. It was then Ligated with the HindIII- BamHI fragment of M13TG889 containing the prepro sequence of the aLpha pheromone in the BglII sites of plasmid pTG848. A new plasmid, referred to as pTG1850, was obtained (Figure 8b).

e e In summary, plasmid pTG1850 possesses: ,15 1) a fragment of the 2 A plasmid of S. cerevisiae which en- S: abLes it to replicate in Saccharomyces; C. 2) the URA3 of S. cerevisiae which enables the transformed 1 cells to be selected from a ura3 receptor strain on medium devoid of a pyrimidine source; 3) the origin of replication of plasmid pBR322 and the gene x for resistance to ampicillin, which enable the plasmid to S* replicate and the transformants to be selected in E. coli; 4) the promoter and the terminator of the yeast PGK gene which provide, respectively, for the initiation and the stopping of transcription of the alpha-amyloglucosidase expression block; '0:0 5) a gene coding for a precursor of this alpha-amyloglucosid- S iase, consisting of the prepro sequences of the alpha pheromone gene (MFalpha

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of S. cerevisiae fused to the sequence coding for the mature portion of alpha-amyloglucosidase which still has its intron D.

Transformation of a Laboratory strain with plasmid pTG1850 DNA pTG1850 plasmid DNA was amplified in E. coli, purified i 35 on a cesium chloride gradient and used f.or transforming the strain TGY2spl3b, which is a haploid strain .of S. cerevisiae i: -having leucine and uracil as.auxotrophic markers (Matalpha, ura3-251-373-328, Leu2 3-12). Following the techniques :I transcviption termination sequence.

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CC Cr CCs described by Ito et at. the ura transformants were tested foar the expression of th e Aspergilus niger amyoglucosidase gene. The technique is as follows: the ura cLones we re subcu Ltured on solid medium c ntaining 2% of starch.

After 48 hours' growth, 5 ml of aqueous iodine mixture (I1 2

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were added to each dish. Tile starch in contact with the iodine (12) takes on a blue coLoration. Yeasts which produce amyloglucosidase in the culture medium degrade the starch and this gives rise to a halo of decoLorization around the .coonies. None of the transformants secretes active amylogI ucosidase.

6) Excision of the intron D by in vitro mutagenesis To carry out this in vitro mutagenesis, an oligonucleotide TG496, the sequence of which is complementary to that of the regions flanking the intron D of phage M13TG1847 DNA, depicted in Figure 9, was hybridized. This oligonucleotide served as a primer for the in vitro synthesis of the strand complementary to the phage M13TG1847 genome. The sequence of the oligonucleotide TG496 is as foLLows: TG496 5' TTCGTCTCTATTGTGGAAACTCACGCCGCA 3'.

The method used is identical to that used for the excision of the intron A. On the other hand, it was not possible to use any restriction enzyme to "sabotage" the nonmutated forms. Several positive signals were observed. The 25 DNA of one of the positive clones was sequenced. The intron D was correctly deLeted. This new phage is referred to as S M13TG1857 (Figure 9).

0 7) Construction of plasmid pTG1858 in the first place, the 670-bp BamHI-SphI fragment of M13TG1857 was isolated and puirified on gel. It was ligated with the large BamHI-SphI fragment of pTG1850. A new amylogucosidase expression vector, referred to as pTG1858, was reconstituted. This vector possesses the region coding for the mature amyloglucosidase of AspergiLLus niger (Figure EXAMPLE 4 STRANSFORMATION OF A LABORATORY STRAIN WITH pTG1858 DNA pTG1858 DNA was used to transform a yeast TGY2spl3b '9 j

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1 13 (MataLpha, ura3-251-373-328, Leu 2-3-18) for ura foLLowing the" techniques described the presence of alpha-gLucoamyLase was tested around the colonies as described above c (Figure 11). As expected, it was found that all the ceLLs transformed with pLasmid pTG1858 secrete active amyLoglucosidase. In addition, the growth of the yeast strains transformed with pLasmid pTG1858 was compared with that of the same strain transformed with pLasmid pTG848. The yeasts transformed with both pLasmids grow on minimaL medium containing 2% of starch as a source of carbon. However, the strain transformed with pLasmid pTG1858 always shows better growth than the strain transformed with pLasmid pTG848 on this medium.

EXAMPLE CHARACTERIZATION OF AN AMYLOGLUCOSIDASE ACTIVITY IN THE YEAST CULTURE

SUPERNATANTS

,I The amount of amyloglucosidase secreted by yeast strain TGY2spl3b containing plasmid pTG1858 was assayed.

After two days' incubation in YNBG medium 0.5% casamino acids at 30°C with stirring at 250 rpm, the culture of STGY2spl3b containing pTG1858 consumed aLL the gLucose and was in stationary phase; the cuLture was arrested at an approximate ceLL density of 2.6 x 10 cells/mL. Measurement of the amount of amytogLucosidase present in the unconcentrated supernatant shows that 360 units of active amyloglucosidase per Liter of supernatant were produced (one unit of amyLoglucosidase activity corresponds to the release of one umol o glucose/min from 2% of ZuLkowsky starch in 50 mM sodium acetate buffer, pH 4.3 at EXAMPLE 6 CONSTRUCTION OF AN INTEGRATION PLASMID FOR SACCHAROMYCES

CEREVISIAE

For this purpose, an EcoRI fragment (1.7 kb) corres- i ponding to the chromosomal region carrying the MFalphai gene of S. cerevisiae was cloned into the EcoRI site of a Sderivative of pUC8 (pTG1864. This new vector is referred V to as pTG1865 (Figure 12).

pTG1865 DN was digested with HindIl, Liberating a aS an i The following statement is a full description o this invention, including the best metTiod of performing it known to me:- 1 'Note: The description is to be typed in double spacing, pica type face, in an area not exceeding 250 mm i; deptii and 10 mm in width, on tough white paper o good qualityand it is to be inserted inside this form.

S11710/76-L iC,, Jlirnhiiariiai ud r Oiinlor, Pcirt 1 "r 14 smaLL, approximately 0.5-kb fragment internal to the MFalphal gene. At the Hindlu sites of pTG1865, there were inserted by blunt-end Ligation the SmaI-BgLII fragment of pTG1858 which includes the amyloglucosidase prepro precursor sequence under the control of the phosphoglycerate kinase gene promoter and the BglII-HindIII fragment of pTG830 which .ncludes the PGK gene terminator sequence, after the action of Klenow polymerase on the HindIII site. This new vector is referred to as pTG1867 (Figure 12). Digestion of pTG1867 with EcoRI liberates a yeast DNA fragment (4 kb) in which the amyLoglucosidase expression block has been integrated,.

EXAMPLE 7 COTRANSFORMATION OF TGY2sp13b FOR THE PURPOSE OF OBTAINING YEASTS SACCHAROMYCES WHICH HAVE INTEGRATED THE AMYLOGLUCO- SIDASE EXPRESSION BLOCK The presence, at the ends of a Linear DNA fragment, S of regions homologous with a region of the yeast genome favors recombination events between the chromosomal region and the linear fragment. This enables us to substitute the sequence of the linear fragment, which can contain foreign sequences, for the chromosomal sequence. It is hence also possible to obtain the integration of foreign sequences in the yeas-t chromosome.

s* The strain TGY2spl3b (Matalpha ura3-251-373-328, leu 2-3-12) was hence cotransformed by the customary technique (14) with 2 Ug of pFL1 DNA conferring the ura phenotype, and at the same time with 10 Ug of pUC1867 DNA which had previously been cut with EcoRI. Of the 300 ura clones obtained, those which had become MFalpha were tested. In effect, the substitution at the MFalphal locus by the linear DNA fragment leads to an inactivation of the MFalpha 1 gene, that is to say a loss in production of the alpha factor of at least The MFalpha 1 phenotype is tested in the following manner: a lawn of FL100 Mata cells (5 x 104 cells) is plated on a dish containing YNBG 0.5% casamino acids. A clump of cells originatintig from the ura clones was deposited on this lawn. After 48 hours at room temperature, the presence of a halo of inhibition of growth of the Mata strain was I B /Y 1 1 11 1 1 1 11 Hence, in the first place, the subject of the pres- 1 ent invention is the expression of AspergillU s niger NCI22343 1 15 observed around the strains having the MFalphal phenotype.

This reflects the fact that the MFalphaI strain secretes sufficient alpha pheromone to inhibit the growth of the Mata strains. No halo of inhibition of growth was observed around the mfalphaI strains. Of the 300 ura clones, one mf-alphai transformant, referred to as TGY2sp13bO APlGB, was obtained. This clone produces a halo of decolorization in the I 2 /KI test after 5 days' growth at 30 0 C. This clone TGY2spl3b 0 A.CB hence produces active amyloglucosidase.

This clone expresses alpha-amyloglucosidase in stable fashion during several generations. Analysis of the genomic DNA of the strain TGY2spl3b 0 AMGB according to Southern reveals that the amyloglucosidase expression block is intega rated in the genome at the MFalphal locus (Figure 13) and that the integration of this foreign fragment has been carried out by an exchange process without integration of the ;vector pTG1864 sequence.

The amount of amyloglucosidase secreted by the yeast.

strain TGY2spl3b 0 AMGB and the strain TGY2spl3b containing plasmid pTG1858 was assayed.

After 2 hours' incubation at 30 0 C with stirring at *a 250 rpm, the strain TGY2spl3b 0 AMGB produces approximately 6- to 8-fold less active amyloglucosidase than the strain *i TGY2spl3b containing plasmid pTG1858.

EXAMPLE 8 INTEGRATION IN A POLYPLOID BREWERY STRAIN SACCHAROMYCES UVARUM (2285TG2) The point at which the amyloglucosidase expression block is integrated in the genome of the brewery strain must be chosen in such a way as not to modify either the physio- Logy or the characteristics of the strain 2285TG2. The choice was made, for the integration site, of the MFalphal gene which is responsible for the synthesis of the alpha factor.

A strain of sex type Mata shows no detectable inhibition of I 35 growth when placed in contact with a polypoid brewery strain.

The alpha factor plays a part in the conjugation between two j; yeasts of opposite sex type. In strain 2285TG2,, inactivation of the MFalphal gene should not modi fy the physiology of.

I .gin of replication in yeast, for exampLe the origin of repli- Scation of the 2 plasnmid.

16 the strain. The presence of this gene in strain 2285TG2 was determined by analyzing the genomic DNA of this strain according to Southern (Figure 14). The MFalpha gene present in the strain 2285TG2 appears to be broadly homologous with that of the S. cerevisiae strain. A single difference was detected in the region coding for the alpha factor situated between the two HindII sites (Figure 12). This region contains, depending on the strain, a variable number of copies of this alpha factor The difference observed is probably due to the presence of at Least one additional copy in the Saccharomyces uvarum strain. In consequence, the amyloglucosidase expression block was inserted into the HindlI sites of the MFalpha I gene of S. cerevisiae bounding the region coding for the alpha factor.

Since the strain 2285TG2 is polyploid and hence has no auxotrophic marker, the cotransformation with pTG1867 is V' accomplished using a plasmid which confers a dominant pheno- Ite type. Resistance to G418 was used. This plasmid which was used is a derivative of plasmid pTG1861 described in French Patent No. 84/04,453. Plasmid pTG1825 differs from pTG1861 c only in respect of the absence of an out-of-phase ATG which v has been deeted n ete n vitro autagenesis in order to permit better expression of the gene for resistance to G418.

Strain 2285TG2 was transformed with 1 gg of pTG1825 DNA and 10 ug of pTG1867 which has previously been digested with EcoRI, byth o, he technique derived from Dickson The following modification was carried out: approximately hours after the transformation, an overlayer of complete medium (10 ml) containing geneticin, 6418 (Difco), was added so that the final concentration of antibiotic was 300 ug/ml in the dish.

l Of 300 G418-resistant clones, one clone, 2285TG2 AMGC, produces amyloglucosidase. This clone expresses alpha-amyloglucosidase in stable fashion during several generations.

Analysis of the genomic DNA of the strain 2285TG2 AMGC accord- S ing to Southern reveals that the amyLoglucosidase expression S block is integrated in the genome at the MFalphal locus. The S amount of amylogLucosidase secreted by the brewery strain was o n l in *e p c ,f c 1 .n 1 u f- h s AT 1 1 1 1 11 1 1 1 1 1 i 1 0 1 1 1 1 1 ,o i i dation of starch to glkucose residucs.

Starting with industrial strains such as Saccharomyces, -17 assayed. After 3 days' incubation in compLete YPG medium at 0 C with stirring at 250 rpm, the 2285TG2 AMGC culture at an approximate ceLL density of 4 x 107 cells/mI produces 100 U of active aLpha-amyLogLucosidase per Liter of supernatant.

EXAMPLE 9 The same experiment as described in Example 8 is carried out starting with a strain used in the bread-making industry.

Since this strain TGF1 is polypLoid and prototrophic, a cotransformation has to be performed with pTG1867 and a pLasmid conferring a dominant phenotype, plasmid pTG1825.

The strain TGF1 is transformed with the DNA of plasmids pTG1825 and pTG1867 which has previousLy been digested with 0~ 00 00 0 EcoRI. Among the 500 cLones resistant to G418 (300 pi mL) obtained, one expresses amyLogLucosidase in stable fashion during at least 50 generations; this cLone is referred to as TGF1-AMG1.

Hybridization of the EcoRI-digested genomic DNAs of C C 0, the strains TGF1-AMG1 and TGF1 with an oLigonucLeotide TG529 specific for the MFalphal gene of Saccharomyces cerevisiae by Southern's technique reveals that the amyLogLucosidase expression block is integrated in the genome at the MFaLphal Locus (Figure 15) of TGF1-AMG1.

The amount of amyLogLucosidase secreted by the strain 0004 TFG1-AMG1 was assayed. After three days' incubation in complete YPG medium at 30 0 C with stirring at 250 rpm, the TGFI- 00; a" AMG1 culture produces 100 U of active aLpha-amyLogLucosidase per Liter of supernatant of a culture at a ceLL density of a 7 5 x 10 ceLLs/mL.

EXAMPLE INTEGRATION OF THE B. LICHENIFORMIS ALPHA-AMYLASE EXPRESSION BLOCK IN THE POLYPLOID STRAIN SACCHAROMYCES UVARUM 2285 TG2

AMGC

,Zv The strain 2285 TG2 AMGC was jtransformed'With 1 g of pTG1825 DNA and 10 ig of M13TG1817 which had previo-usLy een digested with BamHI, by the technique derived from Webter and Dickson (18).D By digestion with BamHI, the vector M13TG1817 V4 t EcoRI. Among i~he 500 cl'ne sitn C1 3 gml the fermentative activity of the baker's yeast to be significantly enhanced and hence the fe-'mentation times to be reduced 18 118 Liberates a DNA fragment containing the His3 gene of Saccharomyces uvarum in which the cassette for expression of aLphaamyLase has been inserted. The construction of the vector M13TG1817 has been described in French Patent No. 86/06,703.

S 5 Of 400 clones resistant to G418 (300 ug/ml), 4 clones produce aLpha-amylase. These clones express alpha-amylase in stable fashion during 50 generations. Hybridization of t the BamHI-digested genomic DNAs of the strains 2285 TG2 AMGC- AMYI and 2285 TG2 AMGC with an oligonucLeotide TG227 specific for the His3 gene of S. uvarum by Southern's technique reveals that the aLpha-amyLase expression block is integrated in the genome at the His3 locus (Figure 16) of the strain 2285 TG2 AMGC-AMY1.

6 The following strains were deposited at the Collection NationaLe de Cultures de Microorganismes (National SCollection of Microorganism Cultures) of the Institut SPasteur, 28 rue du Docteur-Roux 75724 PARIS, Cedex Saccharomyces uvarum 2285 TG2 AMGC on 6th June 1986 under No. 1-568, Saccharomyces cerevisiae TGF1 AMG1 on 27th January 1987 under No. 1-650, Saccharomyces uvarum 2285 TG2 AMGC-AMY1 on 27th January 1987 under No. 1-649.

etc c II 1 i J 1 1 1 1 1 1 I' *1 1 1 1 1 1 111 1 1 i. 1 1 l l l l l o 113, iyr zar ion OT rne :COKL relstrl icion fragments of the genomic DNA of the strains TGY~spl3b AMGB .4 REFERENCESI 1. Brake, Merryweather, Coit, HeberenU.A., Mariarz, Multenbach, G.T.,.Urdea, M.S., Valenzuela, P. and Barr, P.J. (1984) Proc. Nati. Acad. Sci.

USA 81, 4642-4646.

2. Bussey, Saville, Greene, D. et al. (1983) 'Mol.

Cell. Biol. 3, 1362-1970.

3. Grunstein M. and Hogness, D.S. (1975) Colony hybrid-, ization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. NatI. Acad. Sci. USA 72, 'O, 4961-3965.

4. a) BoeL, Hansen, Hjort, Hoegh, 1. and F iii, N.P. (1984) Embo Journal vol. 3, 7, 1581-1585, b) BoeL, Hjort, Svensson, Norris, Norris, K.E. and FiiL, N.P. (1984) Embo Journal vol. 3, 5, 1097-1102.

Langford, C.M. and Gallwitz, D. (1983) Cell 33, 519- 527.

6. Chang, Nunberg, Kaufman, R.M.J. ErLich, Schimke, R.T. and Cohen, S.N. (1978) Nature 275, 617- 624.

7. L Boc, Y, Deyer D. JaeerF., inox, M an 00 *0 Sondermeyer, P. (1986) FEBS vol. 196, 1, 108-112.

8. ZoLLet, M.J. and Smith, M.H. (1983) Meth. in Enzym.

100, 469.

9. Beggs, J. (1981) Genetic Engineering 2, 175-203.

Broach, J.R. (1981) in the toLecuLar Biology of the Yeast Saccharomyces. Life cycLe and Inheritance CHS press, 0:00.: New York.

11. Bach, Lacroute, F. and Botstein, D. (1979) Proc.

NatI. Acad. Sci. (USA) 76, 386-390.

12. SutcLiffe, J.G. (1978) Proc. NatI. Acad. Sci. USA 75 3737-3741.

13. Hitzeman, Hagie, MayffLick, J.S. et at.

(1982) Nucleic Acids Res. 10, 7791-7808.

14.' Ito, Fukuda, Murata, and Kimura, A. (1983) J. Bacteriol. 153, 163-168.

ChevatLier, Block, J.C. and Lacroute,,F. .(1980) Gene 11, 11-19.

.1 which hybridizes with only one of the three oligonucLeotidleS (TG282), corresponds to the 3' region of the AspergiLLus niger 16. Kurjan, J. (1985) MOlecular and Cellular BioLogy 787-796.

17. B rake, Julius,,D'.S. and rhorner, (19,83) Molecular and cellularrBiology 3, 1440-1450.

18. Webster, T.D. and Dickson, R.C. (1983) 26, 243-252.

4 C

Claims (21)

1. 2. The expression block as claimed in claim 1, wherein the DNA sequence which provides for the transcription of transcrip he gene contains the PGK gene promoter.
2. 3. The expression block as claimed in claim 1 or claim 2, which is integrated in Sa plasmid containing an origin of replication in yeasts.
3. 4. The expression block as claimed in claim 3, wherein the origin of replication in yeasts is the origin of replication of the 2 g plasmid.
4. 5. The expression block as claimed in claim 3 or claim 4, wherein the plasmid contains, in addition, a selection gene.
5. 6. The expression block as claimed in claim 5, wherein the selection gene is a gene complementing an auxotrophy. S7. The expression block as claimed in claim 5, wherein the selection gene is a gene conferring a dominant phenotype.
6. 8. The expression block as claimed in claim 7, wherein the dominant phenotype is resistant to G418.
7. 9. The expression block as claimed in any one of claims 1 to 3, which is integrated in the genomepcf the yeast strain. The expression block as claimed in claim 9, wherein the integration is i p frformed in the MFalpha, gene. l' *1 ~or e 1 1 I o oigonucLeotide TG453, TG454 designed, to reconstitute the end of the mature amyloglucosidase and which permit the 0 22
8. 11. A transformed yeast strain containing an expression block as claimed in any one of claims 1 to
9. 12. The strain as claimed in claim 11, which is a strain of Saccharomyces.
10. 13. The strain as claimed in claim 12, which is a strain chosen from S. cerevisiae and S. uvarum.
11. 14. The transformed strain as claimed in any one of claims 11 to 13, which is a polyploid and protrophic strain of Saccharomvces. The strain as claimed in claim 14, which is a strain of Saccharomyces used in the bread-making industry.
12. 16. The strain as claimed in claim 15, which is Saccharomvces cerevisiae strain i I S-- ;TGF1. S17. The strain as claimed in any one of claims 14 to 16, wherein the amyloglucosidase expression block is derived from plasmid pTG1867.
13. 18. The strain of Saccharomvces as claimed in any one of claims 11 to 13, which Sc c C is transformed simultaneously by an expression block as claimed in any one of claims 1 to 10 and by an alpha-amylase expression block, and which simultaneously secretes alpha-amylase and amyloglucosidase.
14. 19. The strain as claimed in claim 18, which is a brewery strain of the C 'I Saccharomvces uvarum type. The strain as claimed in claim 19, which is S. uvarum strain 2285 TG2 AMGC.
15. 21. The strain as claimed in any one of claims 18 to 20, wherein the alpha-amylase t expression block is derived from the vector M13TG1817. t ,L 4 C 22. A process for producing alpha-amyloglucosidase, wherein a strain as claimed in any one of claims 11 to 21 is fermented on a suitable culture medium and wherein the enzyme is recovered in the extracellular medium. S23. A process for panary fermentation, wherein at least one of the strains used in the fermentation is a strain as claimed in any one of claims 14 to 17.
16. 24. A fermentation process, wherein at least one of the strains used in the fermentation is a strain as claimed in any one of claims 18 to 21. A block for the expression of an amyloglucosidase in yeast and a transformed yeast strain containing said block substantially as hereinbefore described with S reference to any one or more of the examples and/or drawings.h S 26. A process for producin alphaamyloguccosidase substantially as hereinbefore inayoeo lis1 o2 sfretdO ~utbecluemdu. hri ecvrdi heetaelua ei devoid of a pyrimidine source. c) The Large EcoRI (coordinate 0)-SaLI (coordinate 650) F. .I 1 described with reference to any one or more of the examples and/or drawings.
17. 27. A process, for panary fermentation substantially as hereinbefore described with reference to any one or more of the examples and/or drawings. DATED this 22nd day of April, 1991. TRANSOENE S.A. 0 *09 *9 00 0 9009 0000 R0Q S V~tt VV C C t r C 4: C C C C CO WIF, CARTER HENDY" PATnn T -2 TRADEMARK ATTORNEYS pLI~j71 QUEENS ROAD, MELBOURNE, 3004, AUSTRAL. [RANSFORMATION OF A LABORATORY STRAIN WITH PTG1856 DNA pTG 1858 DNA was used to transform a yeast TGY spL3b a -I 1/16 A Ai m zz~ c 1771Z1 lS9bp oE 1 poly A, EcoRI 0 PvuI 4084 Pst 13958, EcoRy 830 TGA 970 ~SalT 1000 a a 0 0 moo* 00 0 0 0000* 0 Rose 5*0 0o 0* 9 06 PvuJI 2415' @0 a *0e 09 *o 0 00 0, *e 0 *000 PvuI 5406 Psr 1 5282, @0 NdeI iria~ I''":ia i 1 -a i -:1 i B ii :r Pvui 3741 FIGJA 0 cLi: I, 'in ro as pb1O i ilgure I Z) pTG1865 DNA was digested with HindUl, Liberating a 4;' 21 /16 FIG.2, TTCGTCGCCTAATGTCTCGTCCGTTCACAAACTG AAGAGCTTGIAAGTGGCGAGATGTCTCTGCAG.GAAT CI EcoRi +120 AAGCTAGATGCTAAGCGATATTG CAT IGGCAATAT IGTGTT 90 GATGCATGTGCTTCTTCCTTCAGCTTCCCCTCGTGCGAGTGA GGTTGGC I ATAAA7 TGAAGTGGTTGGTCGGGGTTC CGTGAGGGGCTGAAGTGCTTCCTCCCTTTTAGGCGCAAC TGAGAGCCTGAGCT'TCATCCCCAGCATCATTACACCTCAGCA 9*8 C CC0. C0.. .0 *00C ATG TCG Met Ser TTC CGA TCT Phe Arg Ser CTA Leu CTC Leu GCC Ala CTG Leu GGC Gly *CTC Leu GTC Val TGC, ACA Zys Thr GGG Gly TTG Leu GCA Ala AAT Ain AGC Ser GTG Val TGG Trp ACT Thr .110 C. C ew ATT Ile TTG Leu .TCC Ser AGC Ser AAG Lys AAC Asn CGC Arg GAA Glu GCG Ala GCG Ala ACC Thr ACC Thr TTG. Leu GTG Val GAT TCA Asp. Ser GCT. COT Ala Arg 1, I a GCC ATC CTG AAT AAC Ala Ile Leu Asn Ash ATC -GOO Ile -Gly GCG GAC GOT Asp -Gly 7'] d this Lawn. After 48 hours at room temperature, tne pr-vbvi-.= of a~haLo of inhibition of growth of the flata strain was 22/16 FIG- 2. GCT Ala GTC Val TGG Trp GTT Val GTG Val GCT Ala TCG Ser AGT Ser GGC Gly GCG Ala GAC Asp ACG Thr TCT GGC Ser Gly ATT CCC AGC Pro Ser GAT AAC CCG Asp Asn Pro GAC T GTATGTTTCGAGCTCAGATTTAGTATGAGTGTGTC. Asp IVS ATTGATTGATTGATGCTGACTGGCGTGTCGTTTGTTGTAG A p. a a *1 AC Tyr TTC Phe 4" a a a at 1 (4 a, 4 i 4 CTC GTC Le u VatI Ot r CGA Arg. AlT lie ATT Ile MAT Asn GAG Glu GTC Val TAO Tyr CTC, Leu GGA Gly AAC Asn CAG Gin TCC Ser AAIG Lys ACC TGG Thr Tr p ACC Thr ACC Thr ACT Thr CTC Leu AGT Ser CGC Arg GTC Val CTC Leu GAC Asp GAT Asp CTC L-3u TCT Ser CTC Leu 100 TCC Ser GAT Asp ACC Thr GGT.- Gly Phe TAC ATC TCC GOC CAG Tyr lie, Ser Ala Gin 0GT ATC AGT AAC CCC Gly 'lie Ser Asn Pro AGO GGO GOT GGT CTC- Ser "Gly, Ala :Gly Leu, 110 GCA Ala 120 TOT'GGT Ser Gly 130 GOT GAA Gly Glu GAT CTG Asp ,Leu ii 23/16 FIG-.2 CCC Pro ACT Thr GGT Gly GGC Gly AAG TTC Lys Phe GGT TCT Gly Ser CCG GCT Pro Ala TTC GGG Phe Gly AAT Asn TG Trp CTG Leu CAG Gin GTC GAT Val Asp GGA CGG Gly Arg AGA GCA Arg Ala TGG CTG GAG ACT Glu Thr COG CAG Pro Gin ACT GCT Thr Ala GCC Ala CGA Arg ATG Met 140 TAC Tyr GAT Asp 160 ATC Ile C, p 0 0eC C *0 CC C 6 S op.. S CC.. C. C s.C 0* i~. 0** CS p. CC C S 5505 *5 C C see C *SeOCe 0 CTT GTATGTTCTCCAC Trp Leu Leu CCCCTTG CGTCTGATCTGTGAOATATG TAGOCTGAC~"TG GTCAG IVS B GAO Asp AAT Asn 170 GGC Gly TAO Ty r ACO Thr AGO ACC Ser Thr TG433j I ATT Ilie TCG Ser GTT Val TAT Ty r TAT Tyr TGG Trp 1190 GTG Val COO Pro GOT Ala CTC Leu CAA Gin GTT V61 AGG Arg TGG Trp GOA Ala AAC Asn AAC Asn ACG Thr GAO Asp CAG Gin GAO Asp 'CTG Leu [V TAO Tyr ACA Thr GGA Gly GTGTGTTTGTTTTATTTTtAATTTOOAAAGA IVs C 200 TGOGCCXGCAGAGCTAAOOCGCGATOCGCAG AT Asp OTO Leu ULOCK 15 irtegrateo in the genome at the MFatPhaj Lotus. The amount of amyLogLucosidase secreted by the brewery strain was V 24/16 FIG-2 TGG Trp ACG Thr, GAA Glu TCG Ser GMA Giu ATT Ilie GGT Gly TCC Ser GAA Glu GCT Ala AGT Ser GTC Val GTG Val GCC Ala AAT Asn CAA Gin TTC Phe GGC Gly CAC His GCG Ala TCG Ser CGC Arg ACG Thr TOT Ser GCC Ala GCC Ala 210 TTC Phe 220 CTT Leu 230 GTC Val TTT Phe GTC Val GGC Gly 00 0 0 G.A 0* P *0*0 41 P TGC Cys ccc Pro TGG Trp GAT Asp ACC Thr CCT Pro CAG Gin C1 AC His GAA Glu ACC Thr AGC Ser CTC Lou GAG Giu cC C Pro AAG LVIS. ATT Ile GGC Gly AGC Ser CTG Leu GCC Ala TGC Cys GAG Giu TCC TGG TG387p CTC TGC Leu Cys AGC TTC Ser Phe CGT TCC Arg Ser GGA AGC Gly Ser GCA TGC Ala Cys TCC CCG 'Ser Pro GT GTA Val~ Vat' TGT Cys TAG Tyr ATT Ile GGC Gly GAT Asp CTG Leu CTG Leu AAG Lys TCT Ser GAG Gin GCC Ala GAC Asp ACC Thr TCC Ser CTC Leu 240 CAG Gin 250 TCC Ser 260 AAC Asn' 270 GCA Ala 280 TTT Phe 290 ACC Thr GC Ala 310 CGC Arg. GCA Ala TTC Phe TTc Phe AAC Asn GAT Asp TTC Phe AAC Asn TCA Ser ATC CAC Ilie His GAG Asp GAC Asp CGC. GCG Arg ,Ala GAC TCT TTC Asp Ser ,Phe i ster and Dickson (18). By digestion with BaMHI, the vector M13TG1817 A' 25/16 FIG.2, ATC lie AGC Ser TAT Tyr GAG Glu ACC Thr ,GCT Ala CTc Leu GTT Val ,AC Tyr 'AAC Asn GAT Ash GGT Gly GGT Gly CTC Leu 320 AGT GAC Ser Asp GCG GTG Ala Val CGG Arg 330 TAC Tyr CCT Pro GAG GAO ACG Glu Asp Thr TAO Tyr AAC GGC Asn Gly 340 ~AC COG TGG Asn Pro Trp TG283 *1 .e o 0 0 0 *000 0 0*00 *09's *09 6 S. #1 (I4~ 6 C I I~CC I It I I I *1 *6 I I I 11(6. I It C C I~ ~et C C C C C C C C TTC Phe TTG Leu CAG Gin CTG Leu CTG Leu TAC Tyr GGG Gly GAO Asp TGO Cys GAT Asp TOG Ser TTC Phe ACC Thr GOT Ala TTG Leu TTC Phe TTG, Leu OTA Leu GAG Glu AAG Lys GCT Ala. TAO Ty r GTC Val GCA Ala GC Ala CAG Gin ACA Thr OTG Leu GCA Ala TGG Trp GAT Asp TAO Ty r 350 GAG Glu GAO Asp 370 GTG Val AGO Ser CAG Gin AAG Lys TOG, Ser GAT Asp 390 GCT GCT ACT GGO ACC TAO TCT TCG TOC AGT Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser 400 TOG ACT TAT AGT AGO ATT GTA GAT GOC GTG Ser Thr Tyr Ser Ser lie Vat Asp Ala Val 410 AAG ACT TTOC GC~ GAT GG(C -TOC GTC TCT ATT Lvs Thr Phe Ala Asp Gly Phe Vat Ser Ile GTG GTAAGTCTACGCTAGACAAGCGCTCATGTTGACAG. Val IVS D 26/16 FIG-2 AGGGTGCGTACTAACAGAAGTAG GAA Glu ACT' Thr CAC His GCC Ala GCA AGC AAC Ala Ser Asn TG496 Re *0 S S S~ Re S S C See. 0 S'S. *ee C S so.. U UI4~ Ut t C C C to e* C Re.. *S 0 4 S 440556 U GAC Asp CGC Ar4 CTG Leu GTG Val AGC Ser TCT Ser MAG LYS GAC Asp ACC Thr CT Pro AGC Ser GCC Ala ACC Thr GOC Gly TCT Ser CTG Leu GCC Ala GCT Ala GTG Val AUT Ilie TCG Ser ACC Thr 420 GGC Gly 430 GAT Asp 440 ACC Thr 450 AAC Asn 460 TCT Ser 470 Ccc Pro 480 GGT Ply 490 TGG Trp 500 ACT TCC Ser GGC Gly TGG Trp AAC Asn TGG Trp GGC Gly ACC Thr CCG Pro ATO Met GAG Glu TCT Ser CGT Arg GGC Gly ACC Thr TAC Tyr AGT Ser TCC Ser CAG GIn TAT Tyr CGT Arg GAG Glu TGT Cys AGO Ser ATC Ilie OCT Ala GAG Glu CTT Leu GCT Ala AAC Asn ACC Thr GCG Ala AGT Ser GTG Val ACC Thr TOO Ser GOT Ala TCC Ser TCT Ser GCC Ala GTG Val GOT cc Pro CAA GIn TAC Tyr GCT Ala CTG Leu GTC Val GCC Ala ACA Thr GTC Val GGGC Gly ACI Thr ACT Thr ACT Thr 14 [Nj ACG ACG Thr Thr Thr 0 LI Chevattier, BLock, J.u. ano3 LdtrFUULf, F Gene 11, 11-19. p 27/16 FIG-. GGA Gly ACC Thr TCC Ser ACC Thr GGC Gly GCG Ala 510 AGO Ser 520 ACT Thr GTG Vat GCT Ala ACC Thr ACC Thr AGO Ser TCC Ser TCG Ser MAG Lys TGT Cys 530 AIGT ACG TCA TCA Ser Thr Ser 'Ser ACC Thr. ACC Thr ACC Thr GAT Asp AAC Asn AGC Ser. AGO Ser ACT Thr CTG Leu ATC lie MAG Lys ACC Thr cc Pro ACA Thr TAO Tyr S I 0 00 01 I S S 0*lI 0054 I 04 (t( #4 r C (C 4 4 0* I 05 0 0 4*50 4 45 4 4 4 4 4>14*44 4 4 AC Thr GCT Ala CTG Leu TGG Trp GOT Ala TGG Trp GAG Glu GC Ala ACC Thr GTC Val GAA Glu GTG Val AC Thr GGA Gly ACC Thr 540 GCT Ala 550 ACC Thr TOG Ser 570 AGC Ser GTG Vat TAO Ty r ATO lie GAO Asp ACT Thr GGC Gly TOT Ser GGO Gly. TTO- Phe GAG Glu CAG CTG Gin Leu ATA GOT SIe Ala rG284 GGT GAC Gly Asp 1 GAO Asp TAT Tyr TOG Ser AAG Lys GTC Vat TTT Phe 580 TAO ACT Tyr h 590 ACT GTG Thr Vat U TOO AGIC Ser Ser ACT OTG Thr Leu MAG TTT Lys, Phe GAC Asp CCG Pro ATO Ile OTG Leu CG Pro GOT Ala CGO Arg AGT Ser CTC Leu GGT Gly 600 GAG Glu TAO Tyr 28/16 FIG..2 ATT lie GAG, Glu AGC Ser GAT Asp AAC Asn 610 GAO Asp 620 CGA Arg TCC Ser GTG Val GAG Glu TGG Trp GTT Val GAG Glu AGT Ser GAT Asp CCC Pro GAA Glu TAC Ty r ACC Thr CT Pro a. a. a a a. a. a a a a alit 4 a a a. a. a a.. a. *0 a a.. a a. at a a a. a. a a a a. a a a a a GAG Gin GCG Ala TGC Cys GGA Gly 630 ACG Thr TOG Ser ACO Thr GOG Ala ACG Thr GTG Val ACT Thr GAC Asp 640 ACC TGG CGG TGA CAATCAATCCATTTCGCT Thr Trp Arg Stop ATAGTTAAAGGATGGGGATGAGGGCAATTGGTTATATGATCATG TG 282 TATGTAGTGGGTGTGCATAATAGTAGTGAAATGGAAGOCA *AGTCATGTGATTGTAATCGA CCGACGGAATTGAGGATATO EcoRV CGGAAATACAGACACCGGG 28/16 FIG..2 ATT lie AGT Ser CAG Gin GAG Glu GAT Asp GCG Ala PAGC Ser ccc Pro TGC Cys GAT Asp AAC Asn GGA Gly 610 GAO Asp 620 CGA Arg TCC Ser GAA Gtu GTG Val TAC Ty r ACC Thr GAG TG Glu Trp ACC Thr GCG Ala GTT Vat ACG Thr GAG Gtu COT Pro GTG Val 0@ 00 0 000 0 00 00 0 0 0 0000 0 *000 0004 0 630 ACG TOG Thr Ser 640 ACT GAC ACC TGG CGG TGA CAATCAATCCATTTCGCT a ttThr Asp Thr Trp Arg Stop ATAGTTAAAGGATGGGGATGAGGGCAATTGGTTATATGATCATG TG282 00 0 0 *000 00 0 0 000 0 000.40 0 TATGTAGTGGGTGTGOATAATAGTAGTGAAATGGAAGCCA ,AGTCATGTGATTGTAATCGA CCGACGGAATTGAGGATATC EcoRV CGGAAATAOAGACACCGGG '2 2'. The expression block as claimed in claim 9, wherein the integrjation is ptrformed in the MFalpha, gene. 0 3/16 00 -z q- a a a *f'O a. S S PR St.. a~ a a A *a P tS a r a a B~ *n e a a. a I *f~S S V S t'4P 4~ a SA a IN 033 ]IPV- Cr) M OD q- JOTO Cn (1) AVAI11 1~4 -r reference to any one or more of the examples and/or drawings. 26. A process for producing alpha- amyloglucosidse substantially as hereinbeflore- ft 4/16 '-I uJ '-4 I I r ii ii S S S OS 50 S C S 5505 *555 *555 S 444 S. 4 tv4~ 44 4 50 55 5 4 55 S S 555 5 S S go LL I I~m I A A C A fi a 0 a A Eco RI MI13Tg 1835: P. sI* Il C B A 1111111111A 5- Yll 17 777- ACGGATAACCCGGACTACTTCTACACCT- 3' 3'......GGGTCGTGCCTATTGGGCCTG GAAGATGTGGACCTGAG... AT ATA Sac M413T 9 1837: BamHI Eco RI Avail Bs3HII I II m C" C -1) ii z r- D- ma 0~ Ca LIII a.. a. C C. C S S 'Co a 0. 0 0 .0 a a a.. a a a a a a a a S S *0 a*s S S -a a a a a a Ml3Tg 1839 MI3T9 1837 z Ala Sei- Leu As A CTc TTGGAT Bss NIl Lys AAG T 9 4 53 T 9 454: If ,AGA AAC CTA TTC GCG Hind II I Hind III el- Pst I P51 I* HindfIl M13T 9 1846: AvalI BssHII I I I I I 11 I I I I I I 28Ubp 380bp FIG-6 F7 7 <r -2'a a n :fs) .~u *c 0 24 *I a 9* a a 4r 2 Ca. E#2 a a a O 2 M113T41846 Hind III e Pst I M13T 9 1843 246 d 640 find III et Hind II G) M I3Tg 1846: EcoRV* Hind 110 HindIII ?srI Psi' Ps II f 7 777777 77J V7111111111117 1376bp 670bp FIG-7 x i i H';I- flo 5 i GCC Ala ATC Ile CTG Leu AAT Asn AAC Asn ATC Ile GGG Gly GCG ,Ala GAG GGT Asp 'Gly 1/ N 8/16 P 4 1,12Ikb Hindlil Urc 3 Hind HI 1, 59kb EcoRt 0,8k"I p. p p p p. S 0 a 0500 0q** S p. o p.. *0 S 0 SS S. p p .FIG8a Hind III B 9 1 11/ BamH I* I -4 a. S S S 0 PSUI Sph I Bam HI Eco R I P&I I FIG.. 1 9/16 BamHI indIII 5al I MIST9 889 0 a a a HindIII EcoRI K I FIG8b TGCGC6CCAGAGCTAACCCGCGATCGCAG 1200 AT Asp CTC Leu L~L /1 10/16 IL Hweg XPIH 00 0 0 I 4 9 d- I14ds- I~weg U0 00 (Jo U0 -q uo U u so ~au oAS0O33 u H wele HrPU!H- I Ald, 0 wag SG S S 555 5 555501 S I OAJO' I41A 49d CAC AAG His Lys GAG Glu OTT GTA Val Val GAC Asp TCT Ser TTC Phe 310 CGC Arg TCA Ser 11/16 S. r~ S *6 L w 4 9 Self S 3 ~S* I ftC Zf~ 4: (S 5)50 S IS S S SOS S C 559 I I U- I GTG Val GTAAGTCTACGCTAGACAAGCGCTCATGUTGACAG. IVS D
18. 111-1-1-.- I I 12/16 c .E c 0 -pr Pro *9 9 9 9.9 9 .9 S. 9 9 9 .9.9 99 9 9*9 t 99 99 9 9* 09 9 9 9999 o E 08 a aa -~c site Hind X 0 99 9 0 999 9 9 **0999 9 pIG 1867 FIG-12 GGC. Gly GGC Gly ACC Thr 500 ACT Thr ACG ACG GCT Thr Thr Ala ACC Thr ccc Pro ACT Thr 13 /16 V- 0 m 0 C L e~ ep S. 0 S S SO.. S 5555 0 55.5. S. *&S S 5* o 5 S. S* S S 5,55 -*-MFo(I 55 S S S S 555555 e S FIG-S13 S. S a a a. a Ca. a a a *a a a a a a a 6 a a a a *Sa a aa. .ea a a a 0* S 0 a 8 a a Sea Saa a a a aS 0 a a a a a S R285 T62 HindlE TGY2 Spl13 B Hind I pfG 1867 Hind I 2285T62 EcoRI TGY 2SpI13 EcoRI pTG 1867 Eco RI EmG 1 -n1 P'. 6 1867 oR 1k 2285 TG EcoRI Hind :ir TGY 2SpI3 B EcoR I- Hind 11 CID0 >CoO /16 FIG-lb 0 4 0 S S t S. B C 4- 4- TGF 1 TGF 1-AMG 1 Eco RI pTG 1867 Eco RI U 16 /16 Fl G- 6 p. ~s .st U go .q 0 0 0 ft I. P*~ 0 0400 00 t *0 0 IC ~p C 0<1 a 0 .4 01.00 t~ 4pI I C C C I I I C A B c 4- A 2285 TG2 AMGC B 2285 TG2 AMGC-AMY1 C M 13TG 1817 FE 1TJA TVALUE OF ATTACHED MALOFFICER COMMONWEALTH OF AUSTRALIA.. Patents Act 1952 61211 APPLICATION FOR A STANDARD PATENT (Combined Form Convention and Non-Convention) wJ 4AWe *TRANS GENE SA,. A Frnc p* CD Henri Regnault, 92400 Courbevoie, '0 hereby apply for the grant of a Standard Patent for an invention entitled RB.IQC1(. FQI.... LO TH EXPRESSION OF AN AMINOGLUCOSIDASE IN A.YEAS S TRANSFORMED YEAST, ENZYME PREPARATION PROCESS. AND which is described in the accompanying Complete Specification. :02. This application is a convention application and is based on the application(s) for a &rike our para,2. patent or similar protection made- jqno-cnvntonin T numbered and Apr il 13, 1987 87 05208 So 3. My/Our address for service is: Care of COWlIE, THOMSON CARTER, Patent Attorneys, of 71 Queens Road, Melbourne, Victoria 3004, Australia. DATED this 1.5.th day June 1987 To The Commissioner of Patents COMMONWEALTH OF AUSTRALIA COWIE, THOMSON CARTER. by: Patent Attorneys for TRANSGENE S.A. COWIE, THOMSON CARTER Patent Attorneys 71 Queens Road, Melbourne, Victoria, 3004, Australia FORM 7 &8 AUSTRALIA PATENTS ACT 1952 Insert name of applicant. Insert title of invention. Insert full namelsl a nd addressles) of person(s) making declaration, If applicant a company, person must be authorised to make declaration. Delete alternatives which do not apply Insert name(s) and acrs s lel o)0f actual invienitois(s). Iner etiso eniteen t ppy Inserwdetilseof ea, picnt is .:*Deeofnesrs PATENT DECLARATION FORM (CONVENTION OR NON-CONVENTION) DECLARATION IN SUPPORT OF APPLICATION FOR A PATENT In support of the application made by RNGINESA 16 tu'e Henri P -,nait P2ACIO rouggPFtJOT, FRANCE for a patent for an invention entitled:BTY7 FflP TRf~fExPRPFqqTnN n AN' ZAMINGGEXIMSIDXSE TN A YEASTZPANSFORL4D -YEAST,, ENZYMIE PREPARATION PROCESS AND_ RE I/ Etienne EISENMANN c/o TRANSGENE S 16 rue Henri Regnault, 92400 COURBEVOTE FRANCE do solemnly and sincerely decl are as follows: 1 I- am eap-cat 4or-the-patent- OR I am authorized by the abovementioned applicant to make this declaration on its behalf. 2. I aeg4~-ai *NagfflalfW LA.3AT-,-- n the artuabl-iweGP-&-f-the4ftyentief Fue aes tJenmee±±es, buuu~hWUEVIIU 67000 STRASBOURG, FRANE Ggpard LOTISON, 109 rtte du Faubu~r HatLIna, 67088 STRASBOURex, PRANGE Yves LEMOINE, 4 rtt des Alisiex-, 07100E 4i/are the actual inventor(s) of the invention and the facts upon which the applicant(s) is/&"e entitled to make the application are as follows:- The Applicant is the Assig~ee of' the said actuial inve~ntonr- 3. The basic application(s) as defined by Section 141 of the Act wa6/were made in the follow- ing country or countries on the following dlate(s) by the following applicant(s) in FRANCE -on JUNE 10 -1986 by TRANSGENE S.A. under number 86 08387 by FRANCE on APRIL 13 _1987 by TRANqC,1PNR iin(9,-r mumb0.r 87 05207 in 'PRANCE -on APRIL. 1 3 -19 87. by TRANSCENE S under number 87 05208 in _on 19 by 4. The basic application(s) referred to in paragraph 3 of this Declaration w&6/were the first application(s) made in a Convention country in respect of the invention the subject of the application. Place and date of Signature. Declared at PARIS this Pq clay of MAY 198.7--- NO ATTESTATION OR SEAL Etienne EISENMANN (MANAGER) f fV, c To: The Com~missioner of Patents, Australia j (12) PATENT ABRIDGMENT (11) Document No. AU-B-74435/87 (19) AUSTRALIAN PATENT OFFICE (10) Acceptance No. 612112 (54) Title EXPRESSION OF AMINOGLUCOSIDASE IN YEAST International Patent Classification(s) (51) 4 C07H021/04 A21D 008/04 C12N 001/16 C12N001/18 C12N 001/19 C12N 009/34 C12N 015/00 C12N 015/56 (21) Application No. :74435/87 (22) Application Date 15.06.87 Priority Data (31) Number (32) Date (33) Country 86 08387 10.06.86 FR FRANCE 8705207 13.04.87 FR FRANCE 8705208 13.04.87 FR FRANCE (43) Publication Date 04.02.88 (44) Publication Date of Accepted Application 04.07.91 (71) Applicant(s) TRANSGENE S.A. (72) Inventor(s) NATHALIE LABAT; GERARD LOISON; MARTINE NGUYEN-JUILLERET; YVES LEMOINE (74) Attorney or Agent COWIE CARTER HENDY 71 Queens Road, MELBOURNE VIC 3004 (56) Prior Art Documents EP 163491 AU 59544/86 C12N 15/00 EP 126206 (57) Claim 1. A block for the expression of a mature amyloglucosidase from Aspargillus Niger in a yeast, which block contains: the amyloglucosidase gene devoid of all or part of its introns; the DNA sequence containing the signals which provide for the transcription of the amyloglucosidase gene by yeast; a coding sequence containing the signal required for the export of the mature amyloglucosidase into the extracellular medium, which coding sequence contains a secretion signal chosen from the alpha pheromone, the killer protein secretion system or a synthetic secretion sequence; and a yeast termination sequence after the amyloglucosidase gene and wherein said yeast termination sequence is a transcription termination sequence. ./2 L u i (11) AU-B-74435/87 -2- 612112 11. A transformed yeast strain containing an expression block as claimed in any one of claims 1 to S8. The strain of Saccharomyces as claimed in any one of claims 11 to 13, which is transformed simultaneously by an expression block as claimed in any one of claims 1 to 10 and by an alpha-amylase expression block, and which simultaneously secretes alpha-amylase and amyloglucosidase. A block for the expression of an amyloglucosidase in yeast and a transformed yeast strain containing said block substantially as hereinbefore described with reference to any one or more of the examples and/or drawings. 26. A process for producing alpha-amyloglucosidase substantially as hereinbefore described with reference to any one or more of the examples and/or drawings. L, -i .i I P/00/011 6121 12A 1,I (41{ L Form PATENTS ACT 1 952-1973 COMPLETE SPECIFICATION (ORIGINAL) FOR OFFICE USE Class: Int. Cl: Application Number: Lodged: **Complete Specification-Lodged: Accepted: Published: Priority: Related Art: Name of Applicant: Address of Applicant: TO BE COMPLETED BY APPLICANT TRANSGENE a French body corporate, of 16 rue Henri Regnault, 92400 Courbevole, France. Actual Inventor: NATHALIE LABAT GERARD LOISON MARTINE NCUYEN-JUILLERET YVES LEMOINE Address for Service: 711 QUELN3 1*lAD WWO1~UFRNI, a004, AUSTRAU.A Complete Specification for the invention entitled: BLOCK FOR THE EXPRESSION OF AN AMINOGLUCOSIDASE IN A YEAST, TRANSFORMED YEAST, ENZYME PRF.PARATION PROCESS AND FERMENTAITON PROCESS The following statement is a full description of this invention, includng zhe best method of performing it known to me:--1- 'Note The description is to be typed in double spacing, pica type face, in an area not exceeding 250 mm iii doepth and, 160 mm in Vvidth, on tough white paper of good qUality and it is to bot Inserted inside this form. 117 10/76-L Goss) trusn%% V,01 Is ri-ornincal h mwvr. ra elie, ri tcr ns ature ofrdecrant( t). To: Tha Conmissioner of Patents, Australia The present invention describes processes by means of which different strains of yeast of the genus Saccharomyces, including the industrial strains which do not have the capa- city to degrade complex carbon-containing substrates such as starch or dextrins, may be caused to secrete a glucoamylase. Starch, which is commonL) used as a source of car- bon in alcoholic fermentation, has to be hydroLyzed before- hand (either chemically or enzymaticalLy) in a separate pro- cess in order to produce substrates which are usable for fer- mentative yeasts. It would hence be desirable to construct, by gene- tic recombination, industrial strains which were capable of synthesizing one or more enzymes for degrading starch to sub- strates which are directly assimilable by these strains. More especially, the objective of the present inven- tion is to construct, by genetic recombination, a strain res- ponsible for brewery fermentation capable of degrading the residual dextrins in the wort so as to produce beers having a low content of sugars. Similarly, the enrichment of the wort with respect to fermentable sugars also enables the production of beers having a high alcohol content to be envisaged. The use of a brewery strain which produces an enzyme responsible for the degradation of dextrins during alcoholic fermentation may prove to be a means of simplifying the brew- ing processes and making them profitable. The preparation of beers having a low content of resi- dual sugars can, in particular, be achieved by adding, at various stages of the brewing process, an Aspergillus niger .*30 glucoamylase (amylo-1,6-glucosidase). This glycoamylase cata- lyses the release of glucose residues from the non-reducing ends of starch or dextrins. This enzyme plays a b;g part in the production of glucose syrups from starch and in the manufacture of other alcoholic drinks. This Aspergillus niger glucoamylase is accepted as an additive in the agri-foodstuffs industries. Hence, in the first place, the subject of the pres- ent invention is the expression of Aspergillus niger NCI22343 i, -i i~ -2 aLpha-amylogLucosidase in a brewery strain. Secondly, one of the advantageous features of the invention is that the transformed brewery strains possess the foreign DNA integrated in a yeast chromosome. When the for- eign DNA is integrated in the chromosome according to the pro- cess described Later, the property of producing aLpha-amyLo- gLucosidase is stable and the strain differs from the start- ing strain only in respect of this new character. The stability of the information can also be achie- ved by constructing a DNA structure known as a minichromosome consisting of three main elements: telomere, centromere and origin of replication. Another subject of the present invention relates, more especially, to a functional DNA block enabling amylo- glucosidase to be secreted from /east, this block containing othe following features: 0 m the amyloglucosidase gene devoid of aLL or part of its in- 0 trons; a DNA sequence containing the signals which provide for the 0..0 20 transcription of the amyloglucosidase gene by yeast; a coding sequence containing the signal required for the export of the mature amyloglucosidase into the extracellular medium; the choice was made of a sc 'etion system specific to yeast, that of the alpha pheromone but other sys- tems could be used Cfor example, the killer protein system or that of a synthetic signal sequence]; 00 finally, the expression block may possess, after the amylo- glucosidase gene, a yeast termination sequence, for example that of the phosphoglycerate kinase gene. Si: 0'30 Preferably, the amyloglucosidase gene will be de- void of all of its introns (cDNA). Among the elements of the sequence which provide for the transcription of the amyloglucosidase gene, promoters should be mentioned, in particular the PGK promoter. This functional block can be integrated in an auto- nomously replicating plasmid containing, for example, an ori- gin of replication in yeast, for example the origin of repli- cation of the 2 i plasmid. -i 3 When such a pLasmid is used, it is advantageous that it should be a shuttle plasmid capable of replicating in a bacterium; for this purpose, it may contain elements for rep- lication in a bacterium, for example E. coli, and a selection gene such as that for resistance to an antibiotic, for example Ampr In general, the plasmid will preferably contain an effective gene for selection in yeasts, for example a gene complementing ?n auxotrophy, such as ura 3 or alternatively a gene conferring a dominant phenotype, for example resis- tance to G418. When the expression block is integrated in the genome of the yeast strain, the integration will be performed in a site which is nonessential for the yeast, for example the MFaLphal gene. The constitution of an integration vector is known and will be described in detail in the examples. The invention also relates to the strains containing an expression block according to the invention, involving either a plasmid or a chromosomal integration. Among the transformed strains, there should be men- tioned, in particular, the strains of Saccharomyces, in parti- cular S. cerevisiae and S. uvarum, in particular the indus- trial strains such as the brewery strains. 25 Finally, the invention relates to a process for pre- 90 paring amyloglucosidase, in particular alpha-amyloglucosidase, wherein a strain according to the invention is fermented on a suitable culture medium and the enzyme is recovered in the extracellular medium. *30 The invention relates, finally, to a process for de- grading starch or dextrins, wherein an industrial medium con- taining starch or dextrins is fermented. Finally, the alpha-amyloglucosidase gene used is pre- ferably that of Aspergillus niger. Lastly, the combined action of amyloglucosidase and a liquefying alpha-amylase leads to a total synergy of degra- dation of starch to glucose residues. Starting with industrial strains such as Saccharomyces, T---'CI 4 in which the genetic information for an aLpha-amyLogLucosidase and aLso that for an endo-aLpha-amyLase (for exampLe, of Bacil- lus Licheniformis) are integrated together in the yeast chromo- somes, the production of ethanoL may be obtained using starch as a source of carbon. Thus, the present invention relates to the introduc- tion of the gene for B. Licheniformis aLpha-amyLase and its expression in a strain of Saccharomyces producing aLpha-amyLo- gLucosidase. The simultaneous expression of amyLogLucosidase and ofaLpha-amyLase shouLd Lead to a synergy of these two enzy- matic activities in the degradation of starch or dextrins to gLucose residues. The present invention reLates, more especiaLLy, to the simuLtaneous expression of aLpha-amyLase and amyLogLuco- sidase in a poLypLoid strain of brewer's yeast, S. uvarum, in particuLar the strain 2285 TG2 AMGC, as weLL as to the use of these strains for preparing aLpha-amyLase. Moreover, a most especiaLLy preferred appLication according to the invention reLates to the transformation of particuLar strains of Saccharomyces, in particuLar the poLy- pLoid and prototrophic strains used in the bread-making indus- try; such a strain may be, for example, S. cerevisiae strain TGF1. .25 These poLypLoid and prototrophic strains have to be cotransformed with a vector as described above and a pLasmid which confers a dominant phenotype. One of the subjects of the present invention is, in effect, the use of aLpha-amyLogLucosidase, in particuLar of 30 AspergiLLus niger, during panary fermentation, since aLpha- amyLogLucosidase, which degrades starch directLy to gLucose, has a beneficiaL effect on panary fermentation since this sugar is directLy assimiLabLe by baker's yeast. The addition of this enzyme enables the baking pro- cesses to be improved by making the dough drier to the touch and hence easier to process by machine, and above aLL enabLes the fermentative activity of the baker's yeast to be signifi- cantLy enhanced and hence the fermentation times to be reduced greatly. i 5 Furthermore, this addition should be the source of innovation by enabling relatively sweet bread-type products to be developed, which is virtually impossible without add- ing an enzyme in a short baking process. The present invention hence relates, in addition, to processes by means of which a bakery strain of the Saccharo- myces cerevisiae type is caused to secrete an amyloglucosidase. In particular, the invention relates to a panary fer- mentation process in which at Least one of the strains used in the fermentation is a strain as described above. The examples below are designed to illustrate other characteristics and advantages of the present invention and will be described with reference to the figures, wherein: Figure 1 shows: a) the structure and the restriction map of the Aspergil- lus niger glucoamylase gene, b) the structure of plasmid pTG1831, Sc) the structure of plasmid pTG1830, 20 Figure 2 shows the sequence of the Aspergillus niger amylo- glucosidase gene, o .5 *25 6 0 *0 0 0o 0 *00* 0 SFigure SFigure SFigure SFigure Figure SFigure a) the b) the SFigure Figure Figure 3 shows the structure of M13TG1839, 4 shows the structure and construction of M13TG1843, 5 shows the structure and construction of M13TG1837, 6 shows the structure and construction of M13TG1846, 7 shows the structure and construction of M13TG1848, 8 shows: structure of plasmid pTG1848, structure and construction of plasmid pTG1850, 9 shows the structure and construction of M13TG1857, 10 shows the structure of plasmid pTG1858, 11 shows the detection of the production of amylo- glucosidase by the strain TGY2sp13 pTG1858 by means of the I2/KI test, Figure 12 shows the structure and construction of plasmid pTG1867, Figure 13 shows the hybridization of the EcoRI restriction fragments of the genomic DNA of the strains TGY2sp13b AMGB :i II- L I 6 and TGY2spl3b with the oLigonucLeotide TG529 corresponding to the 3' portion of the MFalphal gene of S. cerevisiae, S Figure 14 shows the hybridization of the restriction frag- ments of the genomic DNA of the strains TGY2spl3b and 2285TG2 with either the oLigonucLeotide TG529 or the oLigonucLeotide TG170, Figure 15 shows the hybridization of the EcoRI-digested genomic DNAs of strains TGF1-AMG1 and TGF1 with an oLigo- nucLeotide TG529 specific for the MFaLphal gene of Saccharomyces cerevisiae, Figure 16 shows the hybridization of the BamHI-digested genomic DNAs of strains 2285 TG2 AMGC-AMY1 and 2285 TG2 AMGC with an oLigonucLeotide TG227 specific for the His3 gene of S. uvarum. EXAMPLE 1 CLONING OF THE ASPERGILLUS NIGER ALPHA-AMYLOGLUCOSIDASE GENE IN FLASMID pBR322 1 pg of AspergiLLus niger NCI22343 genomic DNA and 0.5 pg of pLasmid pBR322 DNA were completeLy digested with EcoRI and SaLI, and these DNAs were then covaLently Linked using T 4 DNA Ligase. Escherichia coli strain 1106 was then transformed with the Ligation products. The seLection of the cLones carrying an insert corresponding to the aLpha-amyLo- gLucosidase gene was carried out after transferring the re- combinant cLones to a nitroceLLuLose sheet according to the cLassicaL method The recombinant cLones were hybridized with a mixture of three oLigonucLeotides TG282, TG283, TG284, defined on the basis of the sequences aLready pubLished TG282 5' ATAGTTAAAGGATGGGGATGAGGGC 3' 3 0 TG283 5' AGGACACGTACTACAACGGCAACCC 3' TG284 5' CTGGGTGACTGGGAAACCAGCGACG 3' a Two positive cLones were isoLated and contain a dif- ferent EcoRI-SaLI fragment. One of the inserts (2.4 kb), which hybridizes with two of the three oLigonucLeotides (TG283, TG284), corresponds to the 5' portion of the amyLo- glucosidase gene. In contrast, the other insert (1 kb), which hybridizes with onLy one of the three oligonucLeotides (TG282), corresponds to the 3' region of the AspergiLLus niger -I O~ i i it ill 7 amylogLucosidase gene (Figure 2). These new pLasmids are referred to as pTG1830 and pTG1831 (Figure 1). EXAMPLE 2 CLONING OF THE cDNA OF ASPERGILLUS NIGER ALPHA-AMYLOGLUCO- SIDASE INTO BACTERIOPHAGE LAMBDA The sequence of the amyLogLucosidase gene, as weLL as that of the cDNA of Aspergillus niger (BU-1) amyLogLucosidase, has already been published (4a, Comparison of these two sequences reveals the presence of 4 intron regions in the Aspergillus niger (BU-1) amyLogLucosidase G1 gene (Figure 2). With the exception of the intron D, which has a se- quence comparabLe to the consensus sequence TATAAC, an essen- tiaL eLement in the mechanism of excision of introns in yeast it appears that none of the other introns can be excised by yeast in vivo. CLoning of the cDNA of amyLogLucosidase was hence undertaken. S* Aspergillus niger NC122343 messenger RNAs were puri- fied from a cuLture of this fungus on rich medium containing 2% of starch as a carbon source, so as to produce a cDNA cor- responding to amyLogLucosidase by reverse transcription. The extracted mRNAs were added to a reaction mixture containing a dT oLigonucLeotide as a transcription primer. The synthesis of the cDNA was carried out according to the 25 method described by Chang et aL. A cDNA Library was pre- 0 pared in bacteriophage Lambda according to Le Bouc et aL. The bacteriophage Lambda DNA was packaged in vitro in the phage particLes, and the phages thereby formed were used to transduce the Escherichia coLi indicator strain POP101. .30 The AspergiLLus niger NCI22343 cDNA Library was screened for amyLogLucosidase, using as a probe an oLigonuc- Leotide TG433 defined on the basis of the pubLished sequences (4a): TG433 5'GGTGTAGCCATTGTCAAGCAGCCACTGCCC 3' corresponding to the sequence compLementary to the sequence coding for amino acids 163 to 172 of the mature protein (Figure 2). Five positive signaLs were detected. Two of the five L 8 cLones were confirmed by cross-hybridization with another oLigonucLeotide TG387 corresponding to the sequence comple- mentary to the sequence coding for amino acids 226 to 236 of the mature protein (Figure 2): TG387 5'CAGGACGAGCCGACGGCCGTCGCGA 3' the size of the inserts in these two candidates being approxi- mately 800 bp. After analysis with restriction enzymes, one of the two fragments was recloned in an M13 derivative (M13mp8) in order to sequence it. This new vector is referred to as M13TG1839 (Figure The sequence of the cDNA fragment of M13TG1839 coding for amino acids 62 to 319 of the mature pro- tein proved to be identical to that already published (Figure 2). EXAMPLE 3 CONSTRUCTION OF AN ALPHA-AMYLOGLUCOSIDASE EXPRESSION VECTOR FOR YEAST S. CEREVISIAE G 1' Reconstitution of the 3' portion of the Aspergillus Leso niger amyloglucosidase gene *20 PLasmid pTG1830 DNA was digested with PstI and SaLI so as to liberate a 1.2-kb fragment. Similarly, plasmid pTG1831 DNA was digested with EcoRI and SaLI, Liberating a 1-kb fragment. These two fragments were isolated and Ligated with M13mp8 DNA which has been digested with PstI and EcoRI. 25 A new vector, referred to as M13TG1843, is reconstituted (Figure This vector M13TG1843 possesses the 3' part of the amylogLucosidase gene. 2) Reconstitution of the 5' portion of the region coding for the mature amyloglucosidase of Aspergillus Niger '30 a) Removal of the intron A by in vitro mutagenesis In the first place, the BamHI-EcoRI fragment (2 kb) of pTG1830 containing the 5' portion of the amyLoglucosidase gene was transferred into M13Tg127 phage DNA between the BamHI and EcoRI sites. This gave single-stranded M13TG1835 DNA, from which the in vitro mutagenesis was carried out. An oligo- nucleotide TG403 (30-mer), the sequence of which is comple- mentary to those of the regions flanking the intron A, was used as a primer for the synthesis of the DNA, using the single 3_ I 9 strand of M13TG1835 DNA as a template (Figure The se- quence of the oLigonucLeotide TG403 is as follows: TG403 5' ACGGATAACCCGGACTACTTCTACACCT 3' One picomole of singLe-stranded M13TG1835 DNA was hybridized with one picomole of oLigonucLeotide TG403 whose ends are phosphorylated in a reaction mixture which con- tains 10 mM Tris pH 7.5, 10 mM MgCL 2 50 mM NaCl. The mix- ture was heated to 100 0 C for 3 minutes and incubated for 1 hour at room temperature. 1.25 pL of each deoxynucLeotide triphosphate at a con- centration of 5 mM was added to the hybridization mixture in order to obtain a final concentration of 500 pM. 5 units of DNA polymerase I KLenow fragment were then added to one unit of T 4 DNA Ligase; the elongation reaction was continued at room temperature for two hours. After precipitation with S ethanol, the Ligation products obtained were digested with the restriction enzyme Sacl. This enables the frequency of pro- duction of recombinant clones containing the mutated form to be increased. Competent ceLLs of E. coLi strain JM103 were 20 transformed with the digestion mixture. The pLaques derived from this transformation were analyzed by DNA/DNA hybridiza- AS* tion, using as a probe the oLigonucLeotide TG403 depicted above, LabeLLed with 3 P by kinase treatment according to the customary techniques SeveraL positive signaLs were ob- '"25 served. The DNA sequence of one of the clones is identicaL s. to that of M13TG1833 DNA without the intron A sequence. This new phage is referred to as M13TG1837 (Figure b) Construction of the vector M13TG1846 The vector M13TG1837 DNA was digested with BssHII and .*30 Avail. The 380-bp BssHII-AvaII fragment containing the DNA sequence coding for the NH 2 -terminaL region of the mature amyLogLucosidase of Aspergillus niger (amino acids 21 to 151) was isolated (Figure SimilarLy, the 280-bp PstI-AvaII fragment of M13TG1839 was isolated. This fragment contains the cDNA sequence coding for amino acids 151 to 256. These two BssHII-AvaII, AvaII-PstI fragments, with two synthetic oligonucleotides TG453, TG454 designed to reconstitute the end of the mature amyloglucosidase and which permit the i i i I i i 10 in-phase reading of the prepro sequence of the alpha phero- mone foLLowed by the mature sequence of amyLogLucosidase (Figure are ligated in M13mp8 which has been digested with HindIII and PstI. This new vector is referred to as M13TG1846 (Figure 6). 3) Construction of M13TG1848 In the first place, the HindIII-PstI fragment of M13TG1846 (670 bp) containing the sequence coding for amino acids 21 to 246 was isolated. M13TG1843 DNA was digested with PstI and EcoRV and the 1736-kb PstI-EcoRV fragment derived from this digestion was isolated and eLuted from a geL. This fragment contains the 3' portion of the amylo- gLucosidase gene (presence of the intron D) coding for amino acids 246-640. The EcoRV site is situated downstream from *0 the TGA stop codon (approximately 140 bp). The poLyadenyL- *:so ation site is situated at approximately 70 bp upstream from the EcoRV site. These two fragments are Ligated in M13TG131 which has been digested with HindIII and HindII. This new vector is referred to as M13TG1848 (Figure This new vec- tor was used as a source of amyLogLucosidase coding sequence for the secretion vector described below, despite the *0* presence of the Last intron D. 4) Construction of an amyLogLucosidase expression vector S* for yeast S. cerevisiae The starting plasmid is pTG848 (identicaL to pTG849 described in French Patent Application No. 83/15,716, with the exception of the ura3 gene whose orientation is inverse); it consists of the foLLowing fragments (Figure 8a): a) The approximately 3.3-kb EcoRI-HindIII fragment origin- ating from pLasmid pJDB207 The HindIII site corres- ponds to coordinate 105 of the 2 i pLasmid B form, the EcoRI site to coordinate 2243. In this fragment, the 2 p is in the B form This fragment enables the plasmid to replicate autonomously in yeast. b) A HindIII fragment carrying the URA3 gene (11) which en- ables the plasmid to be selected in ura3 strains on medium devoid of a pyrimidine source. c) The large EcoRI (coordinate 0)-SaLI (coordinate 650) i I I I 11 fragment of pBR322 In the PvuII site of this frag- ment, there has been inserted the 510-bp EcoRI-HindIII fragment corresponding to the end of the PGK gene the ends of which fragment have been previously rendered blunt by the action of KLenow in the presence of the 4 deoxyribo- nucLeotides. When joined to the PvuII end of pBR322, the flush EcoRI end of the PGK gene regenerates an EcoRI site. d) The HindIII-SaLI fragment (2.15 kb) of the PGK gene (13). The BgLII-HindIII fragment of M13TG1848 was isolated and eLuted from gel. It was then Ligated with the HindIII- BamHI fragment of M13TG889 containing the prepro sequence of the alpha pheromone in the BgLII sites of pLasmid pTG848. A new pLasmid, referred to as pTG1850, was obtained (Figure 8b). In summary, plasmid pTG1850 possesses: *15 1) a fragment of the 2 1 pLasmid of S. cerevisiae which en- abLes it to repLicate in Saccharomyces; 2) the URA3+ of S. cerevisiae which enabLes the transformed .o ceLLs to be seLected from a ura3 receptor strain on medium devoid of a pyrimidine source; the origin of replication of plasmid pBR322 and the gene for resistance to ampiciLLin, which enabLe the pLasmid to replicate and the transformants to be seLected in E. coLi; 4) the promoter and the terminator of the yeast PGK gene which provide, respectively, for the initiation and the stopping of transcription of the alpha-amyLogLucosidase expression block; 5) a gene coding for a precursor of this aLpha-amyLogLucosid- ase, consisting of the prepro sequences of the aLpha phero- mone gene (MFalphal) of S. cerevisiae fused to the sequence coding for the mature portion of aLpha-amylogLucosidase which still has its intron D. Transformation of a Laboratory strain with pLasmid pTG1850 DNA pTG1850 plasmid DNA was amplified in E. coLi, purified on a cesium chloride gradient and used for transforming the strain TGY2sp13b, which is a haploid strain of S. cerevisiae having leucine and uracil as auxotrophic markers (Matalpha, ura3-251-373-3 2 8 leu2 3-12). Following the techniques Mhft. M. 9 o 9 9 12 described by Ito et aL. the u-a transformants were tested for the expression of the Aspergillus niger amylogluco- sidase gene. The technique is as follows: the ura clones were subcultured on solid medium containing 2% of starch. After 48. hours' growth, 5 mL of aqueous iodine mixture (1 2 /KI) were added to each dish. Tne starch in contact with the io- dine (12) takes on a blue coloration. Yeasts which produce amyloglucosidase in the culture medium degrade the starch and this gives rise to a halo of decoLorization around the coLo- nies. None of the transformants secretes active amyloglucos- idase. 6) Excision of the intron D by in vitro mutagenesis To carry out this in vitro mutagenesis, an oligonu- cleotide TG496, the sequence of which is complementary to that of the regions fLanking the intron D of phage M13TG1847 DNA, depicted in Figure 9, was hybridized. This oligonucleo- tide served as a primer for the in vitro synthesis of the strand complementary to the phage M13TG1847 genome. The se- quence of the oligonucleotide TG496 is as follows: 20 TG496 5' TTCGTCTCTATTGTGGAAACTCACGCCGCA 3'. The method used is identical to that used for the ex- cision of the intron A. On the other hand, it was not pos- sible to use any restriction enzyme to "sabotage" the ion- S mutated forms. Several positive signals were obser' a. The DNA of one of the positive clones was sequenced. Che intron D was correctly deleted. This new phage is referred to as S M13TG1857 (Figure 9). 7) Construction of plasmid pTG1858 In the first place, the 670-bp BamHI-SphI fragment of M13TG1857 was isolated and purified on gel. It was liga- ted with the Large BamHI-SphI fragment of pTG1850. A new amyloglucosidase expression vector, referred to as pTG1858, was reconstituted. This vector possesses the region coding for the mature amyLogLucosidase of Aspergillus niger (Figure EXAMPLE 4 TRANSFORMATION OF A LABORATORY STRAIN WITH pTG1858 DNA pTG1858 DNA was used to transform a yeast TGY2sp13b I 1* 13 (MataLpha, ura3-251-373-3 2 8 Leu 2-3-18) for ura foLLowing the techniques described the presence of aLpha-gLuco- amyLase was tested around the colonies as described above (Figure 11). As expected, it was found that aLL the cells transformed with pLasmid pTG1858 secrete active amylogLuco- sidase. In addition, the growth of the yeast strains trans- formed with pLasmid pTG1858 was compared with that of the same strain transformed with pLasmid pTG848. The yeasts transformed with both pLasmids grow on minimal medium contain- ing 2% of starch as a source of carbon. However, the strain transformed with pLasmid pTG1858 always shows better growth than the strain transformed with pLasmid pTG848 on this medium. EXAMPLE .15 CHARACTERIZATION OF AN AMYLOGLUCOSIDASE ACTIVITY IN THE YEAST CULTURE SUPERNATANTS The amount of amyLogLucosidase secreted by yeast strain TGY2spl3b containing pLasmid pTG1858 was assayed. After two days' incubation in YNBG medium 0.5% casamino 20 acids at 30°C with stirring at 250 rpm, the cuLture of TGY2spl3b containing pTG1858 consumed aLL the gLucose and was in stationary phase; the cuLture was arrested at an approxi- mate ceLL density of 2.6 x 108 ceLLs/mL. Measurement of the amount of amyLogLucosidase present in the unconcentrated 25 supernatant shows that 360 units of active amyLogLucosidase per Liter of supernatant were produced (one unit of amyLo- glucosidase activity corresponds to the reLease of one 4moL of gLucose/min from 2% of ZuLkowsky starch in 50 mM sodium acetate buffer, pH 4.3 at 55 0 c). EXAMPLE 6 CONSTRUCTION OF AN INTEGRATION PLASMID FOR SACCHAROMYCES CEREVISIAE For this purpose, an EcoRI fragment (1.7 kb) corres- ponding to the chromosomal region carrying the MFaLpha I gene of S. cerevisiae was cloned into the EcoRI site of a derivative of pUC8 (pTG1864). This new vector is referred to as pTG1865 (Figure 12). pTG1865 DNA was digested with HindII, Liberating a 14 smaLL, approximateLy 0.5-kb fragment internal to the MFaLphal gene. At the HindII sites of pTG1865, there were inserted by blunt-end Ligation the SmaI-BgLII fragment of pTG1858 which includes the amylogLucosidase prepro precursor sequence under the control of the phosphoglycerate kinase gene promoter and the BgLII-HindIII fragment of pTG830 which includes the PGK gene terminator sequence, after the action of KLenow poLymer- ase on the HindIII site. This new vector is referred to as pTG1867 (Figure 12). Digestion of pTG1867 with EcoRI Liber- ates a yeast DNA fragment (4 kb) in which the amyLogLucosid- ase expression bLock has been integrated. EXAMPLE 7 COTRANSFORMATION OF TGY2sp13b FOR THE PURPOSE OF OBTAINING YEASTS SACCHAROMYCES WHICH HAVE INTEGRATED THE AMYLOGLUCO- 15 SIDASE EXPRESSION BLOCK The presence, at the ends of a Linear DNA fragment, of regions homoLogous with a region of the yeast genome favors S recombination events between the chromosomaL region and the Linear fragment. This enabLes us to substitute the sequence of the Linear fragment, which can contain foreign sequences, for the chromosomaL sequence. It is hence also possibLe to obtain the integration of foreign sequences in the yeast chromosome. The strain TGY2sp13b (MataLpha ura3-251- 3 7 3 3 2 8 Leu 2-3-12) was hence cotransformed by the customary technique (14) with 2 pg of pFL1 DNA conferring the ura phenotype, 1 and at the same time with 10 ug of pUC1867 DNA which had pre- viousLy been cut with EcoRI. Of the 300 ura cLones obtained, those which had become MFalphal were tested. In effect, the substitution at the MFaLphal Locus by the Linear DNA frag- ment Leads to an inactivation of the MFaLphal gene, that is to say a Loss in production of the alpha factor of at Least The MFaLphal phenotype is tested in the foLLowing manner: a Lawn of FL100 Mata ceLLs (5 x 10 ceLLs) is plated on a dish containing YNBG 0.5% casamino acids. A clump of ceLLs originating from the ura+ clones was deposited on this Lawn. After 48 hours at room temperature, the presence of a haLo of inhibition of growth of the Mata strain was I I a t 15 observed around the strains having the MFalphal phenotype. This reflects the fact that the MFaLphai strain secretes sufficient alpha pheromone to inhibit the growth of the Mata strains. No haLo of inhibition of growth was observed around the mfalphal strains. Of the 300 ura clones, one mf-alphal transformant, referred to as TGY2spl3bZ AtlGB, was obtained. This clone produces a halo of decoLorization in the 1 2 /KI test after 5 days' growth at 30 0 C. This clone TGY2spl3b 0 AC8 hence produces active amyLogLucosidase. This clone expresses alpha-amyLoglucosidase in stable fashion during several generations. Analysis of the genomic DNA of the strain TGY2spl3b 0 AMGB according to Southern reveals that the amyloglucosidase expression block is integ- rated in the genome at the MFalphal locus (Figure 13) and that the integration of this foreign fragment has been car- ried out by an exchange process without integration of the vector pTG1864 sequence. The amount of amyloglucosidase secreted by the yeast strain TGY2sp13b AMGB and the strain TGY2spl3b containing plasmid pTG1858 was assayed. After 2 hours' incubation at 300C with stirring at 250 rpm, the strain TGY2spl3b 0 AMGB produces approximately 6- to 8-fold less active amyloglucosidase than the strain TGY2spl3b containing plasmid pTG1858. EXAMPLE 8 INTEGRATION IN A POLYPLOID BREWERY STRAIN SACCHAROMYCES UVARUM (2285TG2) The point at which the amyloglucosidase expression block is integrated in the genome of the brewery strain must be chosen in such a way as not to modify either the physio- logy or the characteristics of the strain 2285TG2. The choice was made, for the integration site, of the MFalphal gene which is responsible for the synthesis of the alpha factor. A strain of sex type Mata shows no detectable inhibition of growth when placed in contact with a polyploid brewery strain. The alpha factor plays a part in the conjugation between two yeasts of opposite sex type. In strain 2285TG2, inactivation of the MFalphal gene should not modify the physiology of 16 the strain. The presence of this gene in strain 2285TG2 was determined by analyzing the genomic DNA of this strain accord- ing to Southern (Figure 14). The MFalpha gene present in the strain 2285TG2 appears to be broadly homologous with that of the S. cerevisiae strain. A single difference was detected in the region coding for the alpha factor situated between the two HindII sites (Figure 12). This region contains, de- pending on the strain, a variable number of copies of this alpha factor The difference observed is probably due to the presence of at Least one additional copy in the Saccharomyces uvarum strain. In consequence, the arrylogLuco- sidase expression block was inserted into the HindII sites of the MFaLphal gene of S. cerevisiae bounding the region coding for the alpha factor. *15 Since the strain 2285TG2 is polyploid and hence has no auxotrophic marker, the cotransformation with pTG1867 is accomplished using a plasmid which confers a dominant pheno- type. Resistance to G418 was used. This plasmid which was used is a derivative of plasmid pTG1861 described in French Patent No. 84/04,453. PLasmid pTG1825 differs from pTG1861 only in respect of the absence of an out-of-phase ATG which has been deleted by in vitro mutagenesis in order to permit better expression of the gene for resistance to G418. Strain 2285TG2 was transformed with 1 pg of pTG1825 DNA and 10 pg of pTG1867 which has previously been digested with EcoRI, by the technique derived from Dickson The following modification was carried out: approximately hours after the transformation, an overlayer of complete medium (10 ml) containing geneticin, G418 (Difco), was added so that the final concentration of antibiotic was 300 4g/ml in the dish. Of 300 G418-resistant clones, one clone, 2285TG2 AMGC, produces amyloglucosidase. This clone expresses alpha-amylo- glucosidase in stable fashion during several generations. Analysis of the genomic DNA of the strain 2285TG2 AMGC accord- ing to Southern reveals that the amyloglucosidase expression block is integrated in the genome at the MFalphal Locus. The amount of amyloglucosidase secreted by the brewery strain was L im .i r- 17 assayed. After 3 days' incubation in complete YPG medium at 0 C with stirring at 250 rpm, the 2285TG2 AMGC culture at an approximate cell density of 4 x 10 ceLLs/mL produces 100 U of active alpha-amyLogLucosidase per Liter of supernatant. EXAMPLE 9 The same experiment as described in Example 8 is car- ried out starting with a strain used in the bread-making in- dustry. Since this strain TGF1 is polyploid and prototrophic, a cotransformation has to be performed with pTG1867 and a plasmid conferring a dominant phenotype, plasmid pTG1825. The strain TGF1 is transformed with the DNA of plasmids pTG1825 and pTG1867 which has previously been digested with o.. EcoRI. Among the 500 clones resistant to G418 (300 pg/mL) obtained, one expresses amyLcglucosidase in stable fashion during at Least 50 generations; this clone is referred to as TGF1-AMG1. Hybridization of the EcoRI-digested genomic DNAs of the strains TGF1-AMG1 and TGF1 with an oligonucleotide TG529 specific for the MFalpha I gene of Saccharomyces cerevisiae by Southern's technique reveals that the amylogLucosidase expression block is integrated in the genome at the MFalphal Locus (Figure 15) of TGF1-AMG1. The amount of amylogLucosidase secreted by the strain TFG1-AMG1 was assayed. After three days' incubation in com- plete YPG medium at 300C with stirring at 250 rpm, the TGF1- AMG1 culture produces 100 U of active alpha-amyloglucosidase per Liter of supernatant of a culture at a cell density of x 107 cells/ml. EXAMPLE INTEGRATION OF THE B. LICHENIFORMIS ALPHA-AMYLASE EXPRESSION BLOCK IN THE POLYPLOID STRAIN SACCHAROMYCES UVARUM 2285 TG2 AMGC The strain 2285 TG2 AMGC was transformed with 1 pg of pTG1825 DNA and 10 4g of M13TG1817 which had previously been digested with BamHI, by the technique derived from Web- ster and Dickson (18). By digestion with BamHI, the vector M13TG1817 18 Liberates a DNA fragment containing the His3 gene of Saccharo- myces uvarum in which the cassette for expression of aLpha-- amyLase has been inserted. The construction of the vector M13TG1817 has been described in French Patent No. 86/06,703. Of 400 clones resistant to G418 (300 pg/mL), 4 clones produce aLpha-amyLase. These clones express aLpha-amyLase in stable fashion during 50 generations. Hybridization of the BamHI-digested genomic DNAs of the strains 2285 TG2 AMGC- AMY1 and 2285 TG2 AMGC with an oLigonucLeotide TG227 specific for the His3 gene of S. uvarum by Southern's technique re- veaLs that the aLpha-amyLase expression block is integrated in the genome at the His3 Locus (Figure 16) of the strain S2285 TG2 AMGC-AMY1. The foLLowing strains were deposited at the CoLLec- 15 tion NationaLe de Cultures de Microorganismes (National o CoLLection of Microorganism Cultures) of the Institut Pasteur, 28 rue du Docteur-Roux 75724 PARIS, Cedex Saccharomyces uvarum 2285 TG2 AMGC on 6th June 1986 under No. 1-568, Saccharomyces cerevisiae TGF1 AMG1 on 27th January 1987 under No. 1-65', Saccharomyces uvarum 2285 TG2 AMGC-AMY1 on 27th January 1987 under No. 1-649. e* 0 5 9 I 9* 9 9 0 .9 0* 0 O S 0 009. *9*9 19 REFERENCES 1. Brake, Merryweather, Coit, Heber- Lein, Mariarz, MuLLenbach, Urdea, M.S., VaLenzueLa, P. and Barr, P.J. (1984) Proc. NatL. Acad. Sci. USA 81, 4642-4646. 2. Bussey, SaviLLe, Greene, D. et aL. (1983) MoL. CeLL. BioL. 3, 1362-1970. 3. Grunstein M. and Hogness, D.S. (1975) CoLony hybrid- ization: A method for the isoLation of cLoned DNAs that con- tain a specific gene. Proc. NatL. Acad. Sci. USA 72, -0, 496 1-3965. 4. a) BoeL, Hansen, Hiort, Hoegh, I. and F i iL N.P. (1984) Embo JournaL voL. 3, 7, 1581-1585, b) BoeL, Hjort, Svensson, Norris, Norris, K.E. and FiiL, N.P. (1984) Embo JournaL voL. 3, 5, 1097-1102. 5. Langford, C.M. and GaLLwitz, D. (1983) CeLL 33, 519-
19. 527. 6. Chana, Nunberg, Kaufman, ErLich, Schimke, R.T. and Cohen, S.N. (1978) Nature 275, 617-
20. 624. 7. Le Bouc, Y D reyer, 0 Jaeger, F B inoux, M and Sondermeyer, P. (1986) FEBS voL. 196, 1, 108-112. 8. ZoLLet, M.J. and Smith, M.H. (1983) Meth. in Enzym. 100, 469. 9. Beggs, J. (1981) Genetic Engineering 2, 175-203. Broach, J.R. (1981) in the MoLecuLar BioLogy of the Yeast Saccharomyces. Life cycLe and Inheritance CHS press, New York. 11. Bach, Lacroute, F. and Botstein, D. (1979) Proc. Nati. Acad. Sci. (USA) 76, 386-390. 12. SutcLiffe, J.G. (1978) Proc. NatL. Acad. Sci. USA
21. 3737-3741. 13. Hitzeman, Hagie, MayffLick, J.S. et aL. (1982) NucLeic Acids Res. 10, 7791-7808. 14. Ito, Fukuda, Murata, K. and Kimura, A. (1983) J. BacteriaL. 153, 163-168. ChevaLLier, BLock, J.C. and Lacroute, F. (1980) Gene 11, 11-19. (TG282), corresponds to the 31 region of the AspergiLLus niger I, 20 16. Kurjan, J. (1985) MOLecuLar and CeLLuLar BioLogy 787-796. 17. Brake, JuLius, D.S. and Thorner, J. (1983) MoLecuLar and ceLLuLar BioLogy 3, 1440-1450. 18. Webster, T.D. and Dickson, R.C. (1983) 26, 243-252. 4 4 *4 *4 4 4 a *44* 4 *44~ 444 444 0~ 4 4 4. 4 4 4*44 44 44 4 4 4 444844 21 P, 21 The claims defining the invention are as follows:- 1. A block for the expression of a mature amyloglucosidase from Asp i Niger in a yeast, which block contains: the amyloglucosidase gene devoid of all or part of its introns; the DNA sequence containing the signals which provide for the transcription of the amyloglucosidase gene by yeast; a coding sequence containing the signal required for the export of the mature amyloglucosidase into the .extracellular medium, which coding sequence contains a secretion signal chosen from the alpha pheromone, the killer protein secretion system or a synthetic secretion sequence; and a yeast termination sequence after the amyloglucosidase gene and wherein said yeast termination sequence is a transcription termination sequence. 2. The expression block as claimed in claim 1, wherein the DNA sequence which provides for the transcription of the gene contains the PGK gene promoter. 3. The expression block as claimed in claim 1 or claim 2, which is integrated in a plasmid containing an origin of replication in yeasts. The expression block as claimed in claim 3, wherein the origin of replication in yeasts is the origin of replication of the 2 pt plasmid. 5. The expression block as claimed in claim 3 or claim 4, wherein the plasmid contains, in addition, a selection gene. 6. The expression block as claimed in claim 5, wherein the selection gene is a gene complementing an auxotrophy. 7. The expression block as claimed in claim 5, wherein the selection gene is a gene conferring a dominant phenotype. 8. The expression block as claimed in claim 7, wherein the dominant phenotype is resistant to G418. 9. The expression block as claimed in any one of claims 1 to 3, which is integrated in the genome of the yeast strain. S 10. The expression block as claimed in claim 9, wherein the integration is S/performed in the MFalpha 1 gene. i *C 9@ S 11. A transformed yeast strain containing an expression block as claimed in any one of claims 1 to 12. The strain as claimed in claim 11, which is a strain of Saccharomyces. 13. The strain as claimed in claim 12, which is a strain chosen from S. cerevisiae and S. uvarum. 14. The transformed strain as claimed in any one of claims 11 to 13, which is a polyploid and protrophic strain of Saccharomvces. The strain as claimed in claim 14, which is a strain of Saccharomvces used in the bread-making industry. 16. The strain as claimed in claim 15, which is Saccharomyces cerevisiae strain TGF 1. 17. The strain as claimed in any one of claims 14 to 16, wherein the amyloglucosidase expression block is derived from plasmid pTG1867. 18. The strain of Saccharomyces as claimed in any one of claims 11 to 13, which is transformed simultaneously by an expression block as claimed in any one of claims 1 to 10 and by an alpha-amylase expression block, and which simultaneously secretes alpha-amylase and amyloglucosidase. 19. The strain as claimed in claim 18, which is a brewery strain of the Saccharomvces uvarum type. The strain as claimed in claim 19, which is S. uvarum strain 2285 TG2 AMGC. 21. The strain as claimed in any one of claims 18 to 20, wherein the alpha-amylase expression block is derived from the vector M13TG1817. 22. A process for producing alpha-amyloglucosidase, wherein a strain as claimed in any one of claims 11 to 21 is fermented on a suitable culture medium and wherein the enzyme is recovered in the extracellular medium. 23. A process for panary fermentation, wherein at least one of the strains used in the fermentation is a strain as claimed in any one of claims 14 to 17. 24. A fermentation process, wherein at least one of the strains used in the fermentation is a strain as claimed in any one of claims 18 to 21. A block for the expression of an amyloglucosidase in yeast and a transformed yeast strain containing said block substantially as hereinbefore described with reference to any one or more of the examples and/or drawings. 26. A process for producing alpha-amyloglucosidase substantially as hereinbefore S 9S S a a ,1 c) The Large EcoRI (coordinate 0)-Sail (coordinate 6 U) 23 described with reference to any one or more of the examples and/or drawings. 27. A process for panary fermentation substantially as hereinbefore described with reference to any one or more of the examples and/or drawings. DATED this 22nd day of April, 1991. TRANSGENE S.A. S S 5.55 .5 5 S *S S 5* S *5 SS CMINWiE, CARE 'HENDY PANT:-3 2!YRMVF' rI< TORNEYS 71 QU ;NS OAD, MELBOURNE, 3004, AUSTRALIA