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[SANET-MG] weapons of mass intoxication

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 wine’s 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 “shuttle” 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!


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 <http://www.cfsan.fda.gov/%7Erdb/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


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


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

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


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