Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-06
2001-04-10
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254200, C435S320100, C435S477000, C435S483000, C536S023700, C536S023740
Reexamination Certificate
active
06214577
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the genetic engineering of cells and to beer brewing.
Technology currently exists for introducing heterologous (i.e., modified or foreign) genes into laboratory strains of yeast of the genus Saccharomyces, particularly
S. cerevisiae.
Two types of plasmid vectors have been used for this purpose, replicating and integrating. Replicating vectors contain an origin of DNA replication that functions in yeast, so that the plasmid is maintained extrachromosomally, as a circular episome. Integrating vectors do not contain such an origin and therefore require insertion into a yeast chromosome to be stably maintained.
Both types of plasmids can be introduced into yeast cells by standard transformation methods. Since successful uptake and establishment of plasmid DNA by competent yeast cells is a relatively rare event (<10
−3
), a selection mechanism is required to allow identification of transformants.
Most commonly, selection is accomplished by introducing auxotrophic mutations into the recipient yeast strain. The commonly used mutations are ura3, leu2, trp1, and his3. The plasmid of interest bears a wild type copy of one of these genes. Since the wild type copy on the plasmid is dominant to the host chromosomal allele, selection for cells that receive the plasmid is easily accomplished on a minimal medium lacking the nutrient that is required by the auxotrophic host cell.
There have also been reports of the use of antibiotic resistance to select transformed cells. Replicating vectors have been described that are based on the sensitivity of most Saccharomyces strains to the commercially available neomycin analog, antibiotic G418. Jimenez et al. (1980) Nature 287, 869; Hollenberg (1982) In
Current Topics in Microbiology and Immunology,
Hofschneider et al., eds. (Springer-Verlag NY); Webster et al. (1983) Gene 26, 243. Webster et al. also describe an integrating plasmid vector which could not be directly selected for by resistance to G418. These vectors contain a gene, called kan
r
, neo
r
, or G418
r
from the bacterial transposon Tn903, and a yeast origin of replication; the bacterial gene is preceded by its native bacterial promoter.
Another replicating vector has been described which contains the gene for resistance to the antibiotic hygromycin B under the control of a yeast promoter. Gritz et al. (1983) Gene 25, 178.
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, CO
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
The invention features a vector including a gene for resistance to an antibiotic otherwise capable of killing a host yeast cell, the gene being transcribed from a yeast promoter sequence or synthetic promoter sequence, the vector being capable of being directly selected for.
A gene heterologous to the host yeast cell (i.e., a non-yeast gene, a modified gene, a gene from a different yeast strain, or a homologous gene from a different chromosomal location) can be inserted into the vector, and the vector used to transform the host cells; transformants are selected on the basis of antibiotic resistance.
In a preferred embodiment, the invention features a replicating vector which includes a gene for resistance to G418, which gene is under the control of a yeast control sequence.
In other preferred embodiments, the heterologous gene encodes an enzyme, e.g., glucoamylase (which enables the generation of glucose from starch by the yeast cell), and the host cell participates in a process, e.g., the production of dough, which employs a product of the metabolism of the cell.
In other preferred embodiments, the antibiotic resistance gene and the heterologous gene are under the control of different promoters, the promoter controlling the heterologous gene preferably being the more highly expressed of the two.
Introduction of genes encoding heterologous enzymes into industrial yeast strains using the vectors of the invention will facilitate the production of such products as alcohol, which ordinarily relies on sugars to feed the yeast. An enzyme such as glucoamylase will enable the yeast to break down starch from inexpensive sources such as tapioca and potatoes to yield glucose, which can be fed on by the yeast. Similarly, bread-making can be made cheaper when starch (flour) rather than sugar is used as the primary energy source.
In another aspect, 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 intention 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 exog
Brusca John S.
Clark & Elbing LLP
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