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[SANET-MG] self cloned wine yeast
ISIS Press Release 08/01/07
'Self-Cloned' Wine Yeasts Not Necessarily Safe
Yeast genetics is more precise, but altering the expression of a single
native yeast gene can change the entire metabolic network in an
unexpected way. Prof. Joe Cummins
A fully referenced version of this report is posted on ISIS members’
Self cloned versus genetically modified yeast
The first genetically modified (GM) wine yeast, and the only one
commercially released so far has been described earlier  (GM Wine
Sold Unlabelled in the United States, this series).
In 2006, FDA designated another wine yeast ECMo01 generally recognized
as safe (GRAS), and this is the second to be released for commercial use.
Saccharomyces cerevisiae ECMo01 was derived from Davis 522, a strain
commonly used in the wine industry, and carries a recombinant genetic
insert composed of three elements, the DUR1,2 gene, a promoter, and a
terminator, each of the three parts derived from a different strain of
Davis 522 actually has its own DUR1,2 gene, which is not normally active
during alcoholic fermentation. The purpose of creating ECMo01 is to
increase the expression of urea amidolyase, which catalyzes the
hydrolysis of urea produced by the wine yeast during alcoholic
fermentation. Urea is a precursor of ethyl carbamate (urethane), a
suspected human carcinogen formed in the wine from the reaction of urea
and ethanol; so hydrolyzing urea should significantly reduce the
potential for the formation and accumulation of ethyl carbamate in the
The DUR1,2 gene under control of the S. cerevisiae PGK1 promoter and
terminator signals was integrated into the URA3-locus of Davis 522. In
vivo assays showed that the GM strain reduced ethyl carbamate in
Chardonnay wine by 89.1 percent. Analyses of the genotype, phenotype,
and transcriptome revealed that the GM yeast is substantially equivalent
to the parental strain . Publications were cited to indicate that
ethylcarbamate is a powerful carcinogen in animal and humans, and there
is general agreement about those findings. Interestingly, mice given
ethyl carbamate and wine had significantly reduced incidence of cancer
compared with mice given ethyl carbamate alone. Wine components other
than ethanol seem to play a role in suppression tumours .
The term self-cloning has been coined to describe genetic modification
by gene transfer within the same species, as in the case of
Saccharomyces cerevisae genes from different strains being incorporated
into the GM strain ECMo01.
The issue of self-cloning arose recently in Japan, where a sake (rice
wine) yeast was modified to enhance flavour by incorporating a mutant
fatty acid synthase gene along with an antibiotic resistance gene. A
counter selection procedure was then used to remove the antibiotic
resistance gene but preserves the fatty acid mutant in the chromosome.
The Japanese government has decided that the sake yeast is a
‘self-cloning organism’ not covered by regulations over GM organisms .
Self-cloning covers a growing class of GM wine yeasts that are under
development to enhance or improve the flavour of wines and distillates.
Yeast genes encoding enzymes synthesizing or degrading esters are
targets of manipulation. The genes made to over-express include alcohol
acetyl-transferase and ethanol hexanoyl- transferase. Additional copies
of the genes introduced into GM strains were driven by the yeast PGK1
promoter and PGK terminator. A dominant selectable marker was a mutant
of the yeast acetolactate synthase gene (ilv2) that provides resistance
to the herbicide sulphmeturon. A cassette containing all of the yeast
genes to be integrated was inserted at the ilv2 locus . Even though
bacteria had been used in preliminary cloning, the modified yeast
contained only yeast genes and in that sense it may be comparable to the
Grapes with high sugar content may produce wines with excessive ethanol
leading to public health problems and in impaired flavour. A champagne
strain of wine yeast was modified using the NADH oxidase gene from
Lactococcus lactis under the control of a yeast glyceraldehyde 3
phosphate dehydrogenase promoter integrated into the yeast URA3 locus.
The transgenic wine yeast consumed the high sugar of the must without
producing excessive ethanol . The anti-oxidant resveratrol is a wine
component of proven health benefit. In a novel approach, a coenzyme-A
ligase gene from hybrid poplar and resveratrol synthase gene from
grapevine were both added to the yeast chromosomes. The coenzyme-A
ligase gene with a yeast alcohol dehydrogenase promoter and
transcription terminator was inserted at the URA3 locus of the wine
yeast. The resveratrol synthase gene under the control of the yeast
enolase promoter and terminator was inserted into the LEU2 locus of the
wine yeast. In that way the yeast biochemical pathway was restructured
for enhanced resveratrol synthesis , presumably to produce a double
whammy anti-oxidant. But is it safe?
Transgenic vs self-cloned yeasts
A recent review listed recombinant wine yeasts produced since 1993 to
“improve” wine quality or technology. Of the 14 recombinant wine yeasts,
seven were transgenic and seven were modifications of the genome using
the genes of wine yeasts . It seems sensible for the wine industry to
present the “self cloned” strains as distinct from the transgenic ones.
Both transgenic and self-cloned require careful safety evaluations,
though of the two transgenic wine is probably the greater concern.
Direct comparisons of self-cloned and transgenic wine strains regarding
their commercial and environmental characteristics have not been
reported so far. However, there is a comparative study of the commercial
Baker’s yeast with a transgenic strain and a self-cloned strain altered
for improved frozen bread dough. The yeasts are made resistant to
freezing by disrupting the acid trehalose gene through transformation
with either a yeast uracil 3 gene or a bacterial gene consisting of a
fragment of a gene specifying antibiotic resistance which were directed
to the yeast trehalose gene by short fragments of the trehalose gene at
each end of the disrupting gene sequence . The freeze-resistant strains
were compared in a contained soil environment to determine if the
self-cloned or transgenic strains survived better in the natural
environment than did the wild type yeast. Both the cells and the DNA of
the self cloned and transgenic strains behaved similarly to the wild
type yeast in the simulated natural environment . This type of
experiment might prove informative in wine yeasts.
In a related development, the developers of GM crop plants have used the
term ‘cisgenic’ to describe genetically modified crops derived from
sexually compatible lines. The developers argued that there was no need
for regulatory approval of cisgenic crops provided they are shown to be
free of foreign DNA . There was strong support for the proposal to
deregulation cisgenic crops from industry representatives .
Molecular geneticists David Schubert and David Williams made cogent
arguments against the unregulated release of cisgenic crops. Cisgenic
plants suffer from practically all the major shortcomings of GM
organisms. Cisgenic plants still require the transformation of cells
with DNA, a process widely documented to result in large-scale
translocations of the plant DNA, and scrambling and fragmentation of the
transgene, with frequent random insertions of the plasmid DNA. In
addition, a cisgenic plant would likely lack rigorous, tissue-specific
expression of the introduced gene, thereby allowing aberrant secondary
modifications of proteins, such as glysosylation, that can cause serious
immunogenic responses in animals. Furthermore, regardless of the
presence of regulatory elements, the pattern and level of gene
expression can vary greatly depending upon its insertion site .
Cisgenic crops should not be confused with self-cloned yeasts. Crop
genetic engineering differs fundamentally from yeast genetic
engineering. Crop genetic engineering is based on illegitimate
recombination while yeast genetic engineering employs legitimate
homologous recombination allowing gene insertions at specific sites in
contrast to the unpredictable, uncontrollable insertions in crop genetic
Nevertheless, even self-cloned yeasts must be subject to rigorous and
comprehensive safety tests, as it has already been demonstrated that
changing the expression of a single gene in yeast can have unexpected
effects. In 1995, Japanese researchers reported that a transgenic yeast
engineered for increased rate of fermentation with multiple copies of
one of its own genes ended up accumulating the metabolite methylglyoxal
at toxic, mutagenic levels .
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