Science in Society Archive

'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

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].

Article first published 08/01/07



References

  1. Cummins J. GM wines sold unlabelled in the United States, ISIS Report, January 2007.
  2. US Food and Drug Administration  CFSAN/Office of Food additive Safety  GRAS Notice No.GRN000175  2006
  3. Coulon J, Husnik JI, Inglis DL, van derMerwe, G. K.,Lonvaud, A.,Erasmus DJ and vanVuuren H. Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate in wine. American Journal of Enology and Viticulture 2006, 57,113-24.
  4. Stoewsand GS, Anderson JL and Munson L. Inhibition by wine of tumorigenesis induced by ethyl carbamate (urethane) in mice. Food Chem Toxicol. 1991, 29(5), 291-5.
  5. Hirosawa I, Aritomi K, Hoshida H, Kashiwagi S, Nishizawa Y and Akada R. Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol. Appl Microbiol Biotechnol. 2004, 65(1), 68-73.
  6. Lilly M, Bauer FF, Lambrechts MG, Swiegers JH, Cozzolino D and Pretorius IS The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast. 2006. 23(9), 641-59.
  7. Heux S, Sablayrolles JM, Cachon R and Dequin S. Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions. Appl Environ Microbiol. 2006, 72(9), 5822-8.  
  8. Becker JV, Armstrong GO, van der Merwe MJ, Lambrechts MG, Vivier MA and Pretorius IS. Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol.  FEMS Yeast Res. 2003, 4(1), 79-85.
  9. Gonzalez-Ramos D, Tabera L and Gonzalez,R. Transgenic wine yeast technology comes of age: is it time for transgenic wine? Biotechnol Lett. 2006 Nov 22; [Epub ahead of print]  DOI 10.1007/s10529-006-9236-y
  10. Ando A, Suzuki C and Shima J. Survival of genetically modified and self-cloned strains of commercial baker's yeast in simulated natural environments: environmental risk assessment. Appl Environ Microbiol. 2005, 71(11), 7750-3.
  11. Schouten HJ, Krens FA and Jacobsen,E. Cisgenic plants are similar to traditionally bred plants: international regulations for genetically modified organisms should be altered to exempt cisgenesis. EMBO Rep. 2006, 7(8), 750-3. 
  12. Giddings LV.  ‘Cisgenic’ as a product designation: letter to editor Nature Biotechnology 2006, 24,1329.
  13.  Schubert,D and Williams,D. ‘Cisgenic’ as a product designation: letter to editor Nature Biotechnology 2006, 24, 1327-28.
  14. Inose T and Murata K. Enhanced accumulation of toxic compounds in yeast cells having high glycolysis activity, a case study on the safety of genetically engineered yeast. Intern J Good Sci and Tech 1995, 30, 141-6.

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