ISIS Report 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’ website. Details
here
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 [1] (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 S. cerevisiae.
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 wine [2].
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 [3]. 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 [4].
‘Self-cloning’ yeasts
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 [5].
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 [6]. 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 sake yeast.
Transgenic yeasts
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 [7]. 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 [8], 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 [8]. 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 [9]. This type
of experiment might prove informative in wine yeasts.
‘Cisgenic’ crops
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 [10]. There was strong support for
the proposal to deregulation cisgenic crops from industry representatives
[11].
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 [12].
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 modification.
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 [14].
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