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Yeast transgene-Application

Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species
currently described (estimated to be 1% of all fungal species). Yeasts are unicellular, although
some species with yeast forms may become multicellular. Yeasts do not form a single taxonomic or
phylogenetic grouping. The term "yeast" is often taken as a synonym for Saccharomyces cerevisiae, but
the phylogenetic diversity of yeasts is shown by their placement in two separate phyla: the Ascomycota
and the Basidiomycota .
Transgenic yeast:

A transgenic yeast is one that contain a gene or genes which has been introduced artificially into
yeast genetic makeup using a set of several biotechnology techniques collectively known as
recombinant DNA technology (rDNA). Genetic engineering, i.e. transformation of yeast cells
with recombinant DNA, became feasible for the first time in 1978.
Why yeast is used in recombinant technology:
Eukaryotic cells must be used to study eukaryotic mechanisms of gene function and regulation.
While eukaryotic genes are initially cloned in bacteria, they are later returned to eukaryotic cells
such as yeast for expression and study. Yeast has advantages for use in the production of gene
products (commercially or for research). In recombinant DNA technology yeast is used because
of following reasons.
1. Yeast vectors can be integrative
2. Can autonomously replicate, allowing genes to be isolated, manipulated, and reinserted
in molecular genetic analysis.
3. They are easy to grow.
4. They are very simple eukaryotes.
5. It is simple to reinsert genes into yeast since they can be made to take up DNA by
transformation and have plasmids for the process.
Bacteria and yeast are not suitable for every purpose. For certain applications, plant or animal
cell cultures must be used.

Application of yeast-transgene:
Following are the application of yeast transgenes.
Cheese production:
Rennet is a mixture of enzymes used to coagulate cheese. Originally it was available only
from the fourth stomach of calves, and was scarce and expensive, or was available from
microbial sources, which often suffered from bad tastes. With the development of genetic
engineering, it became possible to extract rennet-producing genes from animal stomach
and insert them into certain bacteria, fungi or yeasts to make them produce chymosin, the
key enzyme in rennet. Approximately 80% to 90% of commercially made cheeses in the
US and Britain were made using FPC. Today, the most widely used Fermentation-
Produced Chymosin (FPC) is produced either by the fungus Aspergillus niger or
Saccharomyces cerevisiae .

Beer yeast and bioethnol production:

1) Bioethanol is primarily produced by fermentation of sugar or the sugar
components of starch. High ethanol yields from lignocellulosic biomass
from agricultural and agro-industrial residues are dependent upon efficient
hydrolysis of sugar polymers and utilization of all the available sugars
including D-glucose, D-xylose, L-arabinose and other fermentable
compounds. S. cerevisiae, which plays a traditional and major role in
industrial bioethanol production, has several advantages due to its high
ethanol productivity as well as its high ethanol tolerance. Different
strategies have been applied to engineer yeast including the introduction of
initial xylose metabolism by using bacterial isomerases, reductases and
polymerases and xylose transport and mutation in hexokinase gene ., these
changes modified the intracellular redox balance and result in over-
expression of xylulokinase and further metabolism via a pentose phosphate
pathway.

2) CFG1 gene has been inserted in Saccharomyces pastorianus which become
helpful in simplication of the process of fermentation for beers with
reduced quantities of alcohol or calories; stability of taste; reduction of lees
during filtering.










3) Engineered Saccharomyces pastorianus are used to make beer that
contribute to the quality of the foam. The foamy head on a glass of beer
consists of small bubbles containing carbon dioxide gas. The gas is
produced by yeast during fermentation. Proteins formed gather around the
gas, forming the bubbles in the foam.



Wine yeast:
Genetically engineered yeast has been produced for optimisation and effenciency
of the process of fermentation, reduction in the length of fermentation times; stability or
improvement of taste; enhancement of fermentation performance, aimed at the complete
fermentation of sugar, the elimination of sweetness in wine; reduction of the fermentation of
Champagne in the bottle; reduction of carcinogenic materials that arise during fermentation and
control of microbial spoilage .As the sexual part of the yeast biological cycle (sporulation and
mating) is not required for wine making, although it is needed for the yeast to survive the adverse
conditions during the cyclic starvation periods between vintages. IME1 is a nonessential gene
required for yeast to enter into meiosis. The use of new ime1 transgenic wine yeasts that are
unable to start meiosis but demonstrate good fermentation performance as a potential strategy for
industrial applications of GMOs, while avoiding their dissemination in nature. These new sterile
yeast strains have good fitness and appropriate genetic markers and could very much help to
forestall the risk of genetic pollution due to the dissemination of GMOs into nature.

Transgenic yeast in heavy metal degradation:
Heavy metals come in waste water plants from industrial
discharges, storm water etc. Toxic metals may damage biological treatment method being
usually inhibitory to both anaerobic and aerobic processes. However, there are certain genetically
modified yeast strains that result in solubilization, chelation, precipitation and volatilization of
heavy metals. Yeast Metallothionein Gene has been modified in Candida utilis and
Saccromycetes cerevisiae that results in chromium and cadmium removal from waste water and
sewages.

Biological uses of Transgenic yeast:
The green fluorescent protein (GFP) is a protein composed of 238 amino acid
residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to
ultraviolet range. GFP traditionally refers to the protein isolated from the jellyfish Aequorea
victoria the GFP gene has been introduced and expressed in many Bacteria, Yeast and other
Fungi, etc. Expression of GFP can be used as a marker for a particular characteristic. GFP can
also be expressed in different structures enabling morphological distinction.They are useful in
includes the labeling of whole organisms, the subcellular localization of cells and proteins ,the
analysis of tissue specificity of gene expression, and fluorescence-activated cell sorting.

Production of bio-pharmaceutal compounds:
Yeast could also be used to produce mammalian milk k-casein. This macropeptide has various
biological activities and is used as a functional food ingredient as well as a pharmaceutical
compound. Since the 1980s, recombinant biotherapeutics have been produced. The first
recombinant protein expressed in S. cerevisiae was human interferon [Hitzeman et al., 1981] and
followed in 1982 by the synthesis and assembly of the hepatitis B surface antigen (HBAg), the
first genetically engineered vaccine [Valenzuela et al., 1982]. To date HBAg is produced by
different strategies in yeast and sold under a variety of names Ambirix and Twinrix etc. Probably
the greatest value provided by S. cerevisiae recombinant technology is through the production of
human insulin. The production covers approximately half of the insulin needed by the diabetics
world-wide. Anticougulents like hirudin and interleukin has also been produced by transgenic
yeast. Human blood proteins, serum albumin and hydrocorticosones has also been obtained from
transgenic yeast like Saccharomyces cerevisiae

Biomedical Research:
Today, the use of transgenic yeast is undergoing a "rebirth" in both fundamental and applied
research. Yeast also paved the way for the systematic analysis of large and complex genomes by
serving as a test bed for novel experimental approaches and technologies, tools that are fast
becoming the standard in drug discovery research, and yeast has improved our understanding and
facilitated the molecular analysis of many disease genes. As a non-pathogenic model of fungal
pathogens it can be used to identify antifungal targets suitable for drug development and to
elucidate mechanisms of action of antifungal agents [Ma, 2001].

Environmental Technologies:
A gene FLO1that encode lectin like cellwall in S.cerevisiae was fused with pro sequence from
Rhizopus oryzae lipase (prorol) is displayed on the cell surface and is capable of biodiesel
synthesis (methylesters synthesized from natural triglycerides.

Human genome mapping:
Transgenic yeast has been extensively used in mapping genome of complex organism (e.g Homo
sapiens) and in forming gene libraries .Yeast Cosmid Vectors have proven to be very convenient
for cloning and sequencing of large segments of yeast chromosomal DNA. To construct a library
with as complete coverage as possible with as few clones as possible, the cloned DNA fragments
should be randomly distributed on the DNA. The insert size is 35-35kb. Yeast artificial
chromosome (YACs) is hybrid of bacterial plasmid and yeast DNA. Rather than antibiotic
selection YACs has selectable marker which are suitable for host system and are able to grow on
selective media.

Baker yeast:
Fresh bakers yeast consists of approximately 3033 % of dry materials, 6.59.3 % of nitrogen,
40.658.0 % of proteins, 35.045.0 % of carbohydrates, 4.06.0 % of lipids, 5.07.5 % of
minerals and various amounts of vitamins, depending on its type and growth conditions. Only
two GM yeast strains have been officially approved for commercial use in 1990 (bakers yeast)
having 77% increase in protein content and 1994 (brewers yeast), but none has been used
commercially.

Sourdough starters:
Sourdough is a mixture of flour and water, containing yeasts and lactic acid bacteria, used as
starter culture to leaven bread. The use of sourdough has a number of important advantages over
bakers yeast, such as the development of characteristic flavor and texture, as well as extension
of preservation time. Transgenic sour dough starter has been produced but they has not been used
commercially. Disruption of the acid trehalase gene (ATH1) by genetic modification (GM)
improves the freeze tolerance of bakers yeast, which is crucial for frozen-dough baking.

Shortcomes of yeast Transgene application:

Only two GM yeast strains have been officially approved for commercial use i.e. bakers yeast
and brewers yeast, but none has been used commercially.
Cells of more complex eukaryotes carry out certain biochemical processes not found in yeast
(e.g. only animal cells produce antibodies) they cant be used as vectors for larger segments of
genome in all cases.
YACs are not used extensively because 2 or more DNA fragments from separate part of genome
can be integrated in to an individual YAC & also YACs frequently lose parts of the DNA during
propagation.

Conclusion:
Yeasts are unicellular, although some species with yeast forms may become
multicellular. It is simple to reinsert genes into yeast since they can be made to take up
DNA by transformation and have plasmids for the process. Many stains of yeast
transgenes have been produced that are used in making cheese, beer, wine, probiotic
products etc. They are used in biomedical, environmental, biotechnological and
molecular techniques. It is dire need of this hour to pay serious attention to
biotechnological and transgenic products to fulfill the need of food, shelter and clother
for this ever growing population.
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