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Genetically Engineered Wine & Yeasts Now on the Market

December 1, 2005

Prof. Joe Cummins

Genetically Modified Wine Now Being Sold

Genetically modified (GM) wine has been marketed in the United States
for the past two years. Presently, only one modification has been
acknowledged but there are a number of modifications reported in wine
yeast and bacteria and a number of modifications reported for grapes.
The United States Food and Drug Administration (FDA) in 2003 designated
the yeast , Saccharomyces cerevisae strain ML01 to be a substance
generally recognized as safe (GRAS). The strain included a gene for
malolactic enzyme from the bacterium Oenococcus oeni and a malate
permease gene from the fission yeast Schizosaccharomyces pombe .Wine
making consists of alcoholic fermentation employing the metabolic
pathways of yeast and malolactic fermentation to convert malic acid to
lactic acid to reduce the acidity of the wine. Malolactic acid
fermentation is usually achieved using lactic acid bacteria which have a
permease for malic acid. Having the two fermentations in one organism
spares the need for the lactic acid fermentation The yeast ML01 was
modified using a shuttle vector containing a chromosome integration
cassette with genes for malolactic enzyme, malate transporter
(permease), regulatory genes and a sequence directing homologous
recombination at a chromosomal locus (not specified in the FDA report).
Chromosome integration was screened by co-transforming the parent yeast
with the integration plasmid accompanied by a plasmid bearing a
selectable marker for the antibiotic phleomycin, then selecting the
antibiotic resistant yeast for ability to for the ability to produce
lactic acid. After culturing the selected antibiotic resistant lactic
acid producing yeast a phleomycin sensitive lactic acid producing strain
was isolated and found to contain the integrated malolactic-malate
transporter genes (the original phleomycin plasmid did not contain a
gene sequence allowing it to be integrated into the yeast chromosome and
the plasmid was ,therefore unstable and frequently lost from the yeast
cell, (1). The company distributing the GM yeast is Springer Oenologie,
Lesaffre Group, of North America. Springer noted that the malate
transporter gene was controlled by the phosphoglycerate kinase gene
(PGK1) promoter and transcription terminator as was the malolactic gene.
.The recombinant yeast softened the wine¹s mouth feel by decreasing its
acidity. Its use also reduces buttery flavors (diactyl) due to lactic
acid secondary metabolism (2).

Genetic modification of yeast and bacteria differs fundamentally from
modification of plants. The modification of bacteria and yeast is based
on homologous recombination while modification of plants is based on
illegitimate recombination. In plants the recombinant gene insertions
are not precise and disrupt genes that are not specifically targeted
while in yeast gene insertions disrupt genes that are targeted. For
yeast genetic engineering ³shuttle² vectors are used to propagate genes
for insertion in yeast in bacteria, when the vector is purified from the
bacterium it is used to transform yeast. The shuttle vector may
replicate autonomously in the yeast nucleus and express genes equipped
with promoter and terminator genes. The expression vector comes equipped
with a sequence homologous with a yeast chromosomal gene. Recombination
between vector and chromosomal gene, disrupts the target chromosomal
gene and inserts the transgene and frequently a selectable marker into
the yeast chromosome at the target locus (3). Details of the
construction of laboratory strains that are not commercial wine yeasts
have been reported. A laboratory yeast strain was modified using shuttle
vectors for the malolactic gene driven by the PGK1 promoter and
terminator and the Schizosaccharomyces malate transporter gene was
similarly driven by the by the PGK1 promoter and terminator (4). In the
laboratory strain a complementing auxotroph could be used as a
selectable marker while the wine yeast requires an antibiotic resistance
marker or as described below a herbicide tolerance marker. The
Schizosaccharomyces malate transporter and malolactic gene driven by the
PGK1 Promoter and terminator were both born on an integration shuttle
vector also containing the SMR1 herbicide tolerance gene. Isolates with
the two Schizosaccharomyces genes and the SMR1 herbicide tolerance gene
were integrated in the ILV2-SMR1 chromosmal locus to enhance stability
of the yeast modifications (plasmid born genes are not stable even
though they are expressed in yeast) (5). The herbicide sulfometuron was
used in the yeast selections described above, it inhibits branched chain
amino acid sythesis. The yeast gene ILV2 encodes acetolactate synthesis
the target of sulfometuron , it is located on the right arm of
chromosome 13 of Sacharomyces cerevisae(6).In yeast it is possible to
target a modified gene to a particular chromomal locus.

A number of reviews have described a range of yeast genetic medications
poised to be released for commercial application. Among the reviews the
report of Schuller and Casal (7) is comprehensive and the general
information that review contains is given below. The areas of wine
production where GM yeast have been tested include aroma liberating
enzymes, acidity adjustment, glycerol production, volatile phenols,
acetate ester formation, hydrogen sulphite reduction, resveratol
production, ethyl carbamate elimination, antimicrobial enzymes, stress
tolerance, sugar uptake, nitrogen assimilation, agrochemicals resistant,
removal of filter clogging polysaccharides and flocculation. The sources
of genes for cloning included a number of fungi, bacteria and poplar
trees. Saccharomyces cerevisae served as the source for about half of
the constructions. It seems likely that a flood of such GM yeast will
soon be deemed GRAS by FDA.

The term self cloning has been coined to describe the cloning of
Saccharomyces cerevisae genes in Saccharomyces cerevisae. An example
would include disruption of a particuoar undesirable gene by insertion
of another gene from Saccharomyces cerevisae. Recently a Sake (rice
wine) yeast was modified in a two step procedure to enhance the flavor
of the sake and to eliminate the antibiotic resistance sequences.. A
mutant fatty acid synthetase gene promoting flavor was integrated into
the genome of the sake yeast along with an antibiotic resistance
sequence and a counter selection marker that signals the loss of the
resistance sequence and the counter selection marker but preserves the
fatty acid mutant in the chromosome. Using counter selection on media
preventing growth of the yeast cells bearing the antibiotic and counter
selection marker the yeast cells with mutant fatty acid gene alone were
recovered. The Japanese government deemed that the sake yeast to be a
self cloning organism which is not covered by regulations over GM
organisms.

Wine yeasts are unstable and sudden losses in heterozygosity have been
observed. Such abrupt changes in the phenotype of wine yeasts are
commonplace (8). Numerous translocations have been observed uniquely in
wine yeasts and such chromosome rearrangements involving transgenes can
lead to unexpected toxicity in the final product (9).Yeasts cells in
wine were found to be hyperactive in mitotic recombination and that
process contributed to the observed instability of wine yeasts (10).

How much yeast nucleic acid is carried over into wine? Autolysis of wine
yeast leads to ribonucleotides that persit in the wine for at least nine
years and contribute to the flavor of wine (11). The fate of DNA from
yeast (using yeast chitinase gene) and the chlorophyll a/b binding
protein from plants along with microsatellite markers showed that the
large DNA markers were present in must (the starting fermentation
mixture, mainly yeast and grape juice) while the 250 base pair micro
satellites were present in both must and young wine up to six months
(12). Live yeast cells were isolated from two bottled wines, a six year
aged white wine and a red dry wine, origination from two different
locations. The two market wines contain yeast were taken from a sample
of five bottled wines (13). The FDA letter designating wine yeast ML01
to be GRAS indicated that the distributor of GM yeast believed that
final wines were free of yeast and yeast DNA but there was no indication
that data had been gathered to support that conclusion, it seemed to be
a simple belief without any scientific basis.

The dissemination and survival of commercial wine yeast in the vineyard
has been studied to provide data needed to consider approval of GM wine
yeast. A three year study indicated that the strains were mainly
recovered at fairly close proximity to the winery up to 200 meters.
Dissemination of the yeast was largely favored by water runoff. The
commercial strains have fluctuations of appearance and disappearance
from the winery environs (14). A comparison of yeast from former and
modern wineries showed that the genetic and wine making properties of
yeast from a winery abandoned in 1914 differed from yeast isolated in a
modern winery . The genetic characteristics of the yeast in the
abandoned winery persisted for over ninety years (15). A study of wine
jars from the tombs of ancient Egypt showed that S. cerevisiae had been
used in winemaking by at least 3150 BC (16). Regulators should give
thought to the time that GM yeast may persist.

There have not yet been commercial releases of GM grapes but there have
been numerous field test releases of GM grapes in USA which may signal a
deluge of commercial releases. There were 25 field test releases of GM
yeast in USA between 1999 and 2005. The bulk of these releases were to
test grapes resistant to diseases including powdery mildew, Botytis,
Agrobacterium, Clostridium, Xylella, nepovirus and and closterovirus.
There was one application for improved fruit quality whose donor gene
was designated confidential business information (CBI). The disease
resistance genes included antimicrobial peptides determined by synthetic
genes, specifying synthetic peptides. The institutions applying for
release permits included Cornell, California and New York State
Universities and theirs were the majority of applications, others
applying were vinters or wine research companies (17).In Europe, Italy
conducted trials of grape modified with a gene regulating the plant
hormone auxin, Germany tested grapes resisting fungal diseases and
France tested grapes resisting nepovirus (18).Australia has done a
number of field tests of grapes modified for grape fruit color or
quality (19). Most of the tests included antibiotic resistance genes as
selectable markers which would very likely be spread among other
organisms during wine making. Recently, there has been a hiatus in
approvals of GM crops for commercial release, that relatively tiny
approval frequency in the face of a very large number of field trials
suggests that bureaucrats may regard the low frequency of approvals to
be a "log jamb" and for that reason facilitate a flood of new approvals
that will be difficult to evaluate because there has been no warning and
relatively few have the time and ability to carefully evaluate the
numerous releases. It is something that the concerned public should be
prepared for.

In the United States approval of GM plants such as grapes is undertaken
by USDA/APHIS and those reviews provide fairly full information which is
made accessible to the public. FDA alone reviews and approves GM
microbes such as yeast used in food products. Their full reviews
including all required support information does not appear to be readily
accessible and their approval reports , such as the GRAS notice report
on GM wine yeast was more like a public relations release on behalf of
the promoters of GM wine yeast (1). The FDA review did not seem to
consider the environmental and human consequences of marketing and
consuming GM wine. The view that the yeast and its autolysis products
including DNA, RNA, proteins and carbohydrates were somehow lost from
the wine was not supported by scientific evidence, only by the
unsupported beliefs of the promoters and reviewers. The GM wine yeast
did not appear to have been tested for toxicity in animal feeding
experiments nor was the must and finished wine. The FDA review seemed to
be based on faith rather than on science. Recently a Medical Journal ,
The Lancet, pointed out that international faith in the FDA is fast
eroding because approvals are frequently influenced by political
pressure and certainly the approval of wine yeast left fundamental
questions to be answered. It is certainly premature to market GM wine
yeast and since the wines produced using GM yeast are not labeled in
markets it is only prudent to avoid all US wines, so goodbye to Gallo!
The Industrial College of the Armed Forces (USA) indicated that the
"biotechnology industry is a critical element of national power" (21). I
hope that military force is not used to make people drink that GM wine!

References

1.US Food and Drug Administration Center for food Safety and Applied
Nutrition (CFSAN) Office of Food Additive Safety Agency Response Letter
GRAS Notice No. GRN000120 June30,2003
http://www.cfsan.fda.gov/~rdb/opa-g120.html

2.Springer Oenologie ML01The First Malolactic Wine Yeast REF OE828 11/04
http://www.lesaffreyeastcorp.com/wineyeast/ML01%20KH%206-13-05.pdf

3. Sikorski RS and Hieter P. A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics. 1989 May;122(1):19-27

4. Volschenk H, Viljoen M, Grobler J, Petzold B, Bauer F, Subden RE,
Young RA, Lonvaud A, Denayrolles M and van Vuuren HJ. Engineering
pathways for malate degradation in Saccharomyces cerevisiae. Nat
Biotechnol. 1997 Mar;15(3):253-7

5. Volschenk H, Viljoen-Bloom M, Subden RE and van Vuuren HJ.
Malo-ethanolic fermentation in grape must by recombinant strains of
Saccharomyces cerevisiae. Yeast. 2001 Jul;18(10):963-70

6. Falco,S and Dumas,K. Genetic analysis of mutants of Saccharomyces
cerevisiae resistant to the herbicide sulfometuron methyl Genetics 1985,
109,21-35

7. Schuller D and Casal M. The use of genetically modified Saccharomyces
cerevisiae strains in the wine industry. Appl Microbiol Biotechnol. 2005
Aug;68(3):292-304

8. Ramirez M, Vinagre A, Ambrona J, Molina F, Maqueda M and Rebollo JE.
Genetic instability of heterozygous, hybrid, natural wine yeasts. Appl
Environ Microbiol. 2004 Aug;70(8):4686-91.

9. Perez-Ortin JE, Querol A, Puig S and Barrio E. Molecular
characterization of a chromosomal rearrangement involved in the adaptive
evolution of yeast strains. Genome Res. 2002 Oct;12(10):1533-9.

10. Puig S, Querol A, Barrio E and Perez-Ortin JE. Mitotic recombination
and genetic changes in Saccharomyces cerevisiae during wine fermentation
Appl Environ Microbiol. 2000 May;66(5):2057-61.

11. Charpentier C, Aussenac J, Charpentier M, Prome JC, Duteurtre B and
Feuillat M. Release of nucleotides and nucleosides during yeast
autolysis: kinetics and potential impact on flavor. J Agric Food Chem.
2005 Apr 20;53(8):3000-7

12. Leopold, S., Uehlein, N., Kaldenhoff, R. and Schartl, A. Fate of DNA
during must fermentation Acta Hort. (ISHS) 2003, 603:133-134
http://www.actahort.org/books/603/603_15.htm

13. Nisiotou AA and Gibson GR. Isolation of culturable yeasts from
market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for
identification purposes.

Lett Appl Microbiol. 2005;41(6):454-63

14. Valero E, Schuller D, Cambon B, Casal M and Dequin S. Dissemination
and survival of commercial wine yeast in the vineyard: a large-scale,
three-years study. FEMS Yeast Res. 2005 Jul;5(10):959-69.

15. Cocolin L, Pepe V, Comitini F, Comi G and Ciani M. Enological and
genetic traits of Saccharomyces cerevisiae isolated from former and
modern wineries. FEMS Yeast Res. 2004 Dec;5(3):237-45.

16. Cavalieri D, McGovern PE, Hartl DL, Mortimer R and Polsinelli M.
Evidence for S. cerevisiae fermentation in ancient wine. J Mol Evol.
2003;57 Suppl 1:S226-32.

17. Grape Field Test Release Permits Database for the U.S. 2005
http://www.isb.vt.edu/cfdocs/fieldtests3.cfm

18. Grape International Field Test Sources Last checked September 20, 2005

http://www.isb.vt.edu/cfdocs/globalfieldtests.cfm

19. Australia PR-145: Evaluation of transgenes in grapevine No. 3 CSIRO
Plant Industry Horticulture Unit 2001and 2002

http://www.isb.vt.edu/cfdocs/globalfieldtests.cfm

20. Editorial Politics Trumps Science at FDA Lancet 2005 366, 1827

21. The Industial College of the Armed Forces USA Industry Studies 2000
Biotechnology

http://www.ndu.edu/icaf/industry/2000/biotech/biotech.htm

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