Background of the Invention This application is a continuation-in-part of Yocum et al. U.S.S.N. 736,450, filed May 21, 1985. This invention relates to the genetic engineering of cells and to beer brewing.

Beer brewing using yeasts, e.g., members of the genus Saccharomyces, requires the presence of mono-, di-, or tri-saccharides in the fermentation culture medium ("wort"), which the yeasts metabolize in the production of ethanol, C0 ₂ and other metabolites. After yeast fermentation, starches and complex oligosaccharides (those larger than three glucose units) remain soluble but unmetabolized. These oligosaccharides, which are flavorless and colorless, add only to the caloric content of beer.

The production of low starch ("light") beer requires removal of some of the unmetabolized soluble starch and complex oligosaccharides present in the wort that normally remain in the beer after fermentation by yeast. Several methods have been used to reduce the content of starch and complex oligosaccharides in low calorie beer:

1) Passing the wort over an immobilized enzyme, glucoamylase, which is capable of breaking down starch and complex oligosaccharides.

2) Addition of soluble glucoamylase to the wort prior to or during fermentation.

3) Prolonging the mashing process, during which endogenous barley amylases degrade starch. 4) Adding malt flour to the wort during fermentation.

5) Substituting fermentable sugars, such as corn syrup, for various amounts of the starch derived from cereal grains.

6) Diluting the final product with water. Summary of the Invention

In general, the invention features a diploid or greater ploidy yeast cell transformed with DNA encoding glucoamylase, the yeast cell being capable of producing enzymatically active glucoamylase. In preferred embodiments, the yeast cell is diploid, triploid, tetraploid, or aneuploid; the glucoamylase-encoding DNA is introduced via a plasmid capable of integrating into a chromosome of the host yeast cell via a sequence on the plasmid homologous with a region of a chromosome of the host cell; the plasmid is integrated into more than one such homologous region-containing chromosome in the host cell; the glucoamylase-encoding DNA is substantially identical to glucoamylase coding sequences of DNA of the mold Aspergillus niger; and the host yeast cell is a beer brewing strain (most preferably lager) used to brew beer, (e.g., light beer) or is a spirits (e.g., whiskey or fuel ethanol) distilling or bread-making strain.

The plasmids of the invention can be integrated in a way which results in the plasmid DNA remaining substantially intact in the host chromosome, or in a way which results in the jettisoning of unwanted plasmid sequences, e.g., E. coli sequences. In both cases, the plasmid includes a region homologous with a region of the host chromosome. In the jettisonning case, the plasmid, prior to transformation, is linearized (as it is also in the non-jettisonning case) , and the homologous sequence of the host chromosome has a first

and a second end and the plasmid includes a first and a second sequence, respectively homologous with the first and second ends, which sequences are separated from each other by a region of partial non-homology which includes the DNA encoding glucoamylase, a third sequence homologous with the corresponding region of the host chromosome, DNA encoding a selectable trait, and DNA encoding a screenable trait.

In another aspect, the invention features an improved method of transforming diploid or greater ploidy yeast cells with plasmid DNA involving contacting the cells and the plasmid DNA under transforming conditions, plating the cells on a porous support, and then selecting transformants, the temperature of the yeast cells being maintained below 40 C for the entire period during the transforming and selecting steps.

The invention makes possible the use of modified forms of the yeast strains normally used in brewing to degrade complex oligosaccharides to produce low-calorie light beer, obviating the addition of exogenous enzyme or diluents, or the use of additional or longer brewing steps, while preserving the distinctive flavor characteristics of the beer imparted by the brewing strain. The invention also makes possible an increased yield of distilled ethanol from the fermentation of grain or other starch-containing mashes. The invention also reduces the sugar requirement in leavening of bread by yeast.

Other features and advantages of the invention will be apparent from the following description of preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments The drawings will first briefly be described. Drawings

Figs. 1 and 3 are diagrammatic representations of plasmids used in the construction of the plasmid of Fig. 2.

Figs. 2 and 5 are diagrammatic representations of plasmids of the invention.

Fig. 4 is the nucleotide sequence of a 58-base segment of synthetic DNA used in the construction of said plasmid. Plasmid Components

As is mentioned above, plasmids of the invention useful for the transformation of yeast cells for the fermentation of starches in the production of, e.g. light beer, include several components, now discussed in more detail.

DNA Encoding Glucoamylase

The glucoamylase-encoding DNA used to transform the yeast cells of the invention can be derived from many sources; the most preferred DNA is the glucoamylase gene of the bread mold A_ _j _ niger. "Glucoamylase" refers to any exo-enzyme capable of degrading glucose-containing oligosaccharides more than three units in length. As will be described in more detail below, it is not necessary that the enzyme include the entire product of the structural gene which encodes the naturally occurring enzyme; we have shown that a less than complete gene product, encoded by a less than complete structural gene, exhibits glucoamylase activity. In addition, some microorganisms produce more than one form of glucoamylase. For example, A^ niger is known to produce two forms of secreted glucoamylase.

called GI and GII (Boel et al. (1984) EMBO J. 3_, 1097) . Form GI results from the splicing out of four introns at the mRNA level; form GII results from the splicing out of the same four introns plus an addition fifth intron of 169 bases located near the 3' end of the transcript. Regulatory DNA

In order for the glucoamylase-encoding DNA to be adequately expressed in the host yeast cells, transcription of the DNA must be under the control of a promoter sequence which is recognized by the yeast transcriptional machinery. Preferred are promoter sequences isolated from or substantially identical to yeast promoters, e.g. the promoter naturally controlling transcription of the S_^_ cerevisiae triose phosphate isomerase ("TPI") gene.

In addition to a promoter sequence, there is preferably, downstream from the glucoamylase-encoding DNA, a suitable transcription terminator, which is also preferably derived from a yeast cell such as £3^_ cerevisiae, and preferably, but not necessarily, derived from the same gene as the promoter used. Integration Sequence

It is preferred that the vector of the invention be capable of integration into a chromosome of the host yeast cell. This is preferably accomplished by means of a sequence on the vector which is homologous with a sequence (a "target" sequence) of a host chromosome. Preferably, the homologous sequence is a region in which integration will not adversely affect the metabolism and flavor characteristics of the host cell. A preferred target region on the host chromosome

is the homothallism (HO_) gene, which is advantageously large, and is not related to flavor characteristics of the host yeast.

Integration provides stability over many host cell generations in the absence of selection, an important advantage in industrial fermentation processes and brewing; autonomously replicating plasmids can be lost from yeast cells at rates up to 1% to 5% per generation. Selectable Marker

Because transformation of yeast cells with plasmids is a relatively rare event, vectors of the invention preferably contain a DNA region which encodes a selectable marker protein for the identification of transformants. This marker protein can be any protein which can be expressed in host yeast cells and which enables the phenotypic identification of yeast cells which express the protein. Preferred marker proteins are proteins which confer resistance to one or more antibiotics, e.g., antibiotic G418. Transformants are those cells able to grow in the presence of the antibiotic. Host Yeast Cells

The yeast cells transformed and cultured according to the invention are diploid or greater ploidy strains used in beer and ale brewing, or in distilled spirits (e.g., whiskey) and bread making. Generally, S. cerevisiae strains, which are "top fermenting" strains, are used in making ales, while S^ uvarum strains, which are "bottom fermenting" strains, are used in making lager beer, including light beer. Beer and ale brewing strains often are tetraploid, while whiskey and other

distillery, and bread strains, are often diploid. Other industrial strains are aneuploid, i.e., of a ploidy not an exact multiple of haploid.

The yeast strains used in the invention are those which already are capable of metabolizing simple sugars to produce the desired ale, beer, whiskey, other distilled spirit, or bread product with the characteristic flavor of the product, and which only lack, prior to transformation according to the invention, the ability to metabolize complex oligosaccharides and starches. Many suitable yeast strains are publicly available.

As already mentioned, the invention permits the production of light beer or ale without additional steps to remove oligosaccharides. In the case of whiskey and other distilled spirits, and bread, the invention permits the use of lower-cost starting materials, i.e. starch rather than sugar, while retaining the desirable flavor characteristics of the fermenting strain. Plasmid Structure

In Figs. 1-3, the following abbreviations are used for restriction endonuclease cleavage sites: A, Xbal; B, BamHI; Bs, BssHII; E, EcoRI; H, Hindlll; K, Kpnl; L, Bell; M, Smal; P, Pstl; PI, Pvul; PII, PvuII; S, Sail; Sp, Sphl; T, Sstll; U, Stul; X, Xhol. (A) denotes the position of a former Xbal site located about 3 kilobases upstream from the 5' end of the HO gene. This site was destroyed and replaced by a Sail site in the construction of an intermediate vector, pRY253, described in Yocum "Yeast Vector", USSN 736,565, assigned to the same assignee as the present application, hereby incorporated by reference. (PII) represents a former PvuII site similarly lost. In

Figure 1, complete genes or gene fusions are shown by boxes. The abbreviations for the genes are as follows: ampr , ampicillin resistance; G418r, antibiotic G418 resistance; HO, homothallism; CYC1, iso-1-cytochrome c; GAL1,. galactokinase; lacZ, beta-galactosidase; GA, A. niger preglucoamylase; TPI, triose phosphate isomerase. The concentric arrows inside the circles indicate segments of DNA having origins as indicated; no arrow indicates E^ coli origin. Other abbreviations are: kb, kilobase pairs; ori, E_ _j _ coli origin of replication.

Referring to Fig. 1, plasmid pDY3, into which the _9_ niger preglucoamylase gene was inserted, is described in Yocum, _id. pDY3 is composed, beginning at the one o'clock position and moving clockwise, of an Xhol to Stul fragment containing a gene fusion of the yeast GAL1 gene and the E^_ coli lacZ gene; a S al to PvuII fragment including the yeast CYC1 promoter and most of the gene for resistance to the antibiotic G418 from the bacterial transposon Tn903 (the non-essential N-terminal region is not included) ; a PvuII to EcoRI fragment including the _ ^~ __ coli origin of replication from pBR322 and the amp ^r gene for selecting transformants in E_^ coli; and an EcoRI to Xbal fragment of S _ cerevisiae containing the HO gene, including a Kpnl site for the insertion of the A_^ niger preglucoamylase gene. Fig. 2 illustrates pRDlll, which contains that gene. In Fig. 2, the source of all DNA is indicated on the inner concentric circle. Vector Construction The first step was the construction of pDY3 as described in Yocum, ld_. The next step was the isolation of the A_^ niger preglucoamylase gene.

Isolation of the A. niger Preglucoamylase Gene A. niger was grown by shaking 10 spores per liter at 30°C in a medium containing, per liter, 7g Yeast Nitrogen Base (Difco) and 20g Soluble Starch (Fisher) . Mycelium was harvested by filtration after 3 days of growth and total RNA was prepared by the method of Lucas et al. (1977) J. Bacteriol. 130, 1192.

PolyA-containing irtRNA was isolated by two passes over oligo-dT-cellulose and used to construct a cDNA library by the standard method of G-C tailing into the Pstl site of pBR322 (Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The cDNA library was transformed into E. coli strain YMC9. Single colonies from about 25,000 transformants were screened with a

32 P-labeled synthetic 27 base oligonucleotide probe corresponding to a ino acids 259-268 of A_ _j _ niger glucoamylase as published by Svenson et al. (1983)

Carlsberg Res. Commun. 4J^, 529. The sequence of the 27-mer was:

5 ^■ -GCATGCGACGACTCCACCTTCCAGCCC-3' Twelve clones that hybridized with the probe were characterized. One of them, designated pl-19A, contained a 2,200 base pair insert that was shown by DNA sequence analysis to contain the entire coding sequence for preglucoamylase I, as described by Boel et al. (1984) EMBO J. 3_, 1097.

The following constructions and steps are illustrated in Figure 3. A unique Bell restriction site was located 54 base pairs downstream from the terminaton codon of the

preglucoamylase I gene in pl-19A. This site was converted into an Xbal site by standard methods (Maniatis et al. (1982), id) to yield plasmid pRY301 . A BssHII to Pstl fragment containing bases 69-746 of the preglucoamylase I coding sequence was cut out of pRY301 and ligated together with a synthetic 58-mer that replaces sequences lost in the subcloning of the BssHII to Pstl fragment (Fig. 4) into the EcoRI to Pstl backbone of pUC8 (New England Biolabs) to give pRDlOl.

Construction of Glucoamylase Fusion Gene Preglucoamylase was expressed as a fusion protein from the S^ cerevisiae TPI promoter. The gene coding for the fusion protein contains DNA including the TPI promoter, the first three amino acids of TPI, an EcoRI linker which creates an isoleucine codon, and preglucoamylase I beginning at the leucine at the sixth position. The DNA sequence around the fusion junction is: Met Ala Arg lie Leu Leu... ATG GCT AGA ATT CTA CTC

TAC CGA TCT TAA GAT GAG

The staggered line indicates the EcoRI cleavage site at the fusion junction. The gene fusion was constructed as follows. pRY271 is an expression vector containing an EcoRI linker inserted at codon three of TPI and a natural Xbal site just upstream from the TPI transcription terminator (Fig. 3). Between the aforementioned EcoRI and Xbal sites was inserted two DNA fragments, the EcoRI-Pstl piece of preglucoamylase cDNA from pRDlOl, and the Pstl-Xbal piece of preglucoamylase cDNA from pRY301. This yielded pRDl05.

The entire gene fusion from pRDl05, containing the TPI promoter and terminator, and the TPI-preglucoamylase gene fusion on a Smal-HindiII fragment, was then transferred by blunt end ligation with Xhol linkers into the Kpnl site of pDY3 (Fig. 1) , to yield the integrating plasmid pRDlll (Fig. 2) .

An additional plasmid, pRDl33 (Fig. 5) , was constructed which contained a cDNA encoding a protein which mimics GII, described above; construction was as follows. Plasmid pRD105 was cut with Ba HI and Xbal to remove the 3' end of the glucoamylase gene. The resulting fragment was ligated with a synthetic linker of the following sequence: 5' - GATCCTAGTAAC GATCATTGGATC -5', to yield pRDl33. Plasmid pRDl33 was designed to encode a protein that is missing the a ino acids covering the fifth intron, as well as a several additional amino acids on both sides of the intron. The linker was designed such that no extraneous amino acid sequences were introduced.

Upon transformation into yeast, pRDl33 yields a slight increase (about 10%) in glucoamylase activity over pRDl05, so yeast strains containing the shortened version of the glucoamylase gene may be preferred in some instances. The shortened version can be easily transferred to the integrating vector pDY3 on a Smal to HindiII fragment, in a manner analogous to the construction of pRDlll from pRDl05 as described above. An integrating vector containing the shortened version of the glucoamylase gene can also be constructed

from pRDlll as follows. pRDlll can be partially cleaved with BamHI under conditions that give an average of one BamHI cut per molecule. Full length linear plasmid can then be separated from circular (uncut) plasmid by standard preparative gel electrophoresis. The isolated linear plasmid can then be cleaved with Xbal and ligated with the synthetic linker described above. Transformation of Polyploid Brewing Strains

American lager beer strains are more difficult to transform than most other yeast strains. In fact, we found it impossible to transform American lager strains with integrating plasmids using standard procedures such as is described in Webster et al. (1983) Gene J26_, 243. Therefore, we devised a new method that is more efficient than standard procedures and that routinely allows transformation of American lager strains with integrating plasmids such as pRDlll. The new method, which involves, as have previous methods, the use of antibiotic resistance to select transformants, avoids exposing the yeast cells to heated, molten agar, which we have found kills many or all of the cells. Instead, we expose the cells to the antibiotic by plating the cells on a porous support, e.g., filter paper, which is placed on top of solid, cool medium containing antibiotic, which contacts the cells after diffusing up through the porous support. In more detail, the method is as follows.

Lager strains were isolated from kegs of unpasteurized beer, e.g., Budweiser, by filtration of 500 ml beer through a .45 micron Nalgene disposable filter unit. The filter was excised with a sterile scalpel and placed on a petri plate of YEP-D agar (1% Difco Yeast Extract, 2% Difco Bacto-Peptone, 2% dextrose, and 2% agar) containing 20 ug/ml tetracycline

and 100 ug/ml ampicillin. Yeast colonies appeared in three days. The yeast strain was identified as a close relative of Saccharo yces cervisiae by DNA hybridization of 2 micron DNA and HO DNA. For transformation, the lager strains are typically grown to 2 x 10 cells/ml in YEP-D liquid

9 medium. 4 x 10 cells are pelleted by centrifugation

(all centrifugations are 5,000 rpm for 5 minutes) and rinsed once in 20 ml LTE (0.1 M lithium acetate, 0.01 M Tris-HCl, pH 7.4, .001 M Na ₂ EDTA) . The cells are then resuspended in 20 ml LTE and incubated for 30 minutes at 30°C on a roller drum. Cells are then pelleted, resuspended in 2.0 ml LTE, and aliquoted into 0.2 ml portions. 25 ug of plasmid DNA linearized at a site in the target sequence (for example, the unique

Sstll site in HO in the case of pRDlll) is mixed with 25 ug of sheared calf thymus DNA in a total volume of 25 to 50 ul LTE and added to a 0.2 ml aliquot of treated cells. The mix of DNA and cells is kept on ice for 10 minutes and then is heat shocked in a 38°C water bath for 5 minutes. After 10 more minutes on ice, 1.0 ml of 40% Polyethyleneglycol 4000 in LTE is mixed with the cell suspension. After 30 minutes on ice, the cells are pelleted and resuspended in 0.2 ml YEP-D. 0.1 ml of this suspension is spread on a Millipore filter (catalog number HATF 082 25) that has been placed flat on the surface of a YEP-D agar 0.1M KPO., pH 7.0 petri plate. After incubation at 30° for 2 generations (6-8 hours for American lager strains) , the filter containing the yeast cells is transferred to a fresh petri plate of YEP-D agar 0.1M KP0 ₄ , pH 7.0 plus 200-1000 ug/ml antibiotic G418. Care is taken to avoid bubbles of air between the agar and filters. Transformants appear out the background of untransformed cells as colonies after

3 or 4 days at 30°C. This procedure typically gives about 25-50 transformants per 25 ug of linearized integrating plasmid. The integrated state of the plasmid is routinely confirmed by Southern Blot analysis. Jettisonning of Vector Sequences A transformant containing pRDlll integrated at the HO locus is grown for 20-40 generations non-selectively in YEP-D liquid medium and plated at about 500 cells per petri plate on YEP-Gal-XG-BU agar (1% yeast extract, 2% peptone, 2% galactose, 0.006% 5*-bromo-4'-cloro-3*-indoyl-Beta-D-galactoside, 0.1 M KP0 ₄ , pH 7.0, 2% agar). After 5 days at 30°C, most colonies turn blue. Rare white colonies are picked onto MS agar (.7% Difco Yeast Nitrogen Base, 2% Fisher soluble starch, 2% agar) to check for growth on starch as a sole carbon source. About one in 10 3 to 104 colonies are white, and about one in two of the white colonies secrete glucoamylase as evidenced by growth on starch. Confirmation of glucoamylase secretion is routinely checked by Western Blotting and identification of glucoamylase with a rabbit antibody to purified A. niger glucoamylase.

Once the gene encoding glucoamylase has been integrated and the unwanted sequences jettisonned, the above-described procedure may be repeated, to integrate additional copies of the gene into other chromosomes, or other locations within a chromosome. This can result, e.g. , in the integration of the gene into every chromosome of a host yeast cell having the homologous target region. Creating multiple copies of the gene yields these advantages: 1) increased expression of the gene; 2) increased stability of the resulting strains, perhaps by decreasing the probability of gene conversion which could result in the loss of the gene.

Brewing Strains A single copy of the TPI-glucoamylase fusion was deposited in one copy of the IJO gene of an American lager brewing strain. Brew 1, as described above. The vector sequences were jettisoned and the final structure of the deposited gene was confirmed by Southern Blots. This new strain is called Brew 1/pRDlll-R (the R stands for Revertant) .

Two batches of beer were simultaneously brewed from the same lot of wort, one batch with Brew

1/pRDlll-R and the other with untransformed Brew 1. The wort contained, per liter, 150 grams of Munton and Fison Amber Malt Extract, 0.5 gram Hallertau Hops Pellets, 0.5 gram Burton Water Salts, and 2.0 grams of Yeast Nutrient Salts (Beer and Wine Hobby, Greenwood, MA 01880). A 5% innoculu was grown aerobically to saturation in wort and then added to an anaerobic fermentor. After 9 days of fermentation at 15°C, the raw beer was tranferred to a clean fermenter, leaving behind the bulk of the settled yeast, after which the beer was stored for 3 weeks at 15 C.

The fermented beer was then analyzed for the presence of dextrins. A 1 ml sample of beer was treated with 1 ul of a commercial preparation of A. niger glucoamylase (DIAZYME 200L ^R , Miles Laboratories) for 3 hours at 50 C. These conditions had been shown to effect complete digestion of any residual dextrins to glucose. A 25 ul sample of each digest was then analyzed for glucose on a Yellow Springs Instruments Model 27 glucose analyzer. The beer brewed by the transformed strain. Brew 1/pRDlll-R, contained substantially reduced levels of dextrin compared to the control beer brewed by untransformed Brew 1. High pressure liquid chromatography of the two beers, using a 10 cm Spheri 5

RC 8 column coupled to a 22 cm Polypore H column (both from Rainin Instruments) and .01 N H-SO. as the eluant, confirmed that residual dextrins were reduced by the engineered strain compared to the control strain.

Deposit

Plasmid pRDlll in E. coli YMC9 has been deposited in the American Type Culture Collection, Rockville, MD, and given ATCC Accession No. 53123. Applicants' assignee, BioTechnica International, Inc., acknowledges its responsibility to replace this culture should it die before the end of the term of a patent issued hereon, and its responsibility to notify the ATCC of the issuance of such a patent, at which time the deposit will be made available to the public. Until that time the deposit will be made available to the Commissioner of Patents under the terms of 37 CFR §1.14 and 35 §112.

Other Embodiments

Other embodiments are within the following claims. For example, as mentioned above, any suitable diploid or greater ploidy yeast can be used in the invention. Suitable strains include the ones listed below, all of which contain the HO gene, which facilitates integration.

Strain Name Species Type

Budweiser S. uvarum lager

ATCC 42928 S. cerevisiae wine

Fleischman S. cerevisiae bread

Red Star S. cerevisiae bread

Red Star Quick Rise S. cerevisiae bread

1354 S. diastaticus lab

DBY 745 S. cerevisiae lab

DCL-M S. cerevisiae distillery