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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C5/00—Other raw materials for the preparation of beer
  • C12C5/004—Enzymes
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
  • A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
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  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C11/00—Fermentation processes for beer
  • C12C11/003—Fermentation of beerwort
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C12/00—Processes specially adapted for making special kinds of beer
  • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
  • C12C12/006—Yeasts
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  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C12/00—Processes specially adapted for making special kinds of beer
  • C12C12/02—Beer with low calorie content
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/90—Isomerases (5.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• Beer brewing using yeasts 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.
• 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.
• 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:
• 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.
• 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;
• 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
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• a suitable transcription terminator which is also preferably derived from a yeast cell such as £ ⁇ _ cerevisiae, and preferably, but not necessarily, derived from the same gene as the promoter used.
• 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.
• 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.
• 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.
• 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.
• S. cerevisiae strains which are "top fermenting” strains, are used in making ales
• S ⁇ uvarum strains which are "bottom fermenting” strains
• 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.
• 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.
• the invention permits the production of light beer or ale without additional steps to remove oligosaccharides.
• 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.
• 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.
• 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_ ⁇ n
• Fig. 2 illustrates pRDlll, which contains that gene.
• 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
• 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.
• 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.
• 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
• 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.
• 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.
• pRDl33 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
• 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.
• the lager strains are typically grown to 2 x 10 cells/ml in YEP-D liquid
• 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.
• 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.
• 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.
• 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.

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