Hennie JJ van Vuuren (Ph.D.)
Professor and Eagles Chair
Most red wines and some white wines, notably Chardonnay, undergo
bacterial malolactic fermentation (MLF) to decarboxylate malic
acid (tart) in wine to
lactic acid that has a softer mouthfeel. MLF also renders wine
The bacterial MLF process is unreliable despite the availability of commercial
cultures of Oenococcus oeni. Wineries around the world experience problems on
regular basis with this secondary fermentation and sluggish and stuck MLF’s
to spoilage of wines and the production of biogenic amines. Malolactic bacteria
lactic acid bacteria present in fermenting grape must produce toxic biogenic
Lonvaud-Funel, 2001 for a review) such as histamine, cadaverine, phenylethylamine,
putrescine and tyramine (Zee et al., 1983; Lehtonen, 1996, Lonvaud-Funel, 2001).
chemicals in wine have been shown to produce undesirable physiological effects
susceptible individuals; histamine causes headaches, and other allergenic symptoms
as, hypotension, palpitations, flushing, oedema, diarrhea, and vomiting (Wantle
1994; Santos, 1996; Soufleros et al., 1998). Tyramine and phenylethylamine are
associated with migraines and hypertension (Soufleros et al., 1998). Biogenic
also linked to carcinogenesis. Nitrosable secondary amines such as dimethylamine,
piperidine, pyrrolidine, spermidine, spermine in wine can react with nitrous
acid and its
salts to form carcinogenic nitrosoamines (Santos, 1996). Histamine, putrescine,
spermidine and spermine can induce cell transformation and tumour pathogenesis
(Medina et al., 1999; Pryme et al., 2001; Wallace et al., 2001). Use of ML01
wineries to add sulphite at a much earlier stage of fermentation (no more sulphite
required than is usually applied) that will prevent the growth of malolactic
bacteria. Switzerland is the first country to ban the sale of wine containing
exceed a certain concentration. Consumers will benefit directly since wines produced
with ML01 will be free of allergenic bioamines and precursors to carcinogens
by lactic acid bacteria.
The genetic construction (7 years) and testing (7 years) of the malolactic
ML01 is described in Volschenk et al., 1997 (PDF), Husnik
et al., 2006 (PDF) and Husnik
et al., 2007 (PDF). The malolactic wine yeast contains no antibiotic resistance
gene and analyses of the phenotype, genotype, transcriptome, proteome and metabolome
have shown conclusively that ML01 is substantially equivalent to the parental
de Mousse S92. Wineries will benefit from the use of ML01
since this yeast efficiently conducts the MLF, no spoilage of wine will occur
due to stuck MLF, wines have lower
volatile acidity (acetic acid – experts opposed to ML01 have predicted
an increase in
volatile acidity without any data available to them), colour properties of wine
improved, wine quality is higher, wines are more fruity and have an improved
(body) (see Husnik et al., 2007).
The malolactic yeast ML01 is the first genetically improved yeast to receive
approval from the FDA and Health Canada and Environment Canada
application. A Notification has been submitted to the Registrar
Genetic Resources in
South Africa requesting permission for commercial application in South African
wineries. Notifications are currently being prepared for all of the other major
producing regions in the world.
A number of reports have been published on the internet by individuals
opposed to the use of ML01 for the commercial production of wine.
All of these
publications contain faulty information, or deliberate misinformation.
Center for Biosafety and Biowatch SA have objected to the use of
ML01 for the
production of commercial wines in South Africa. Their objections
and our response to
their objections are also provided in this document.
1. Dr. Joe Cummins
Cummins wrote a report in Sustainable Agriculture Research and Education
the internet in which he describes the construction of ML01 and
condemns the use of this
yeast for winemaking http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A2=ind0512&L=sanet-mg&P=6101
Cummins stated that “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)”. We did not
use a shuttle vector to transform the parental yeast strain as claimed by Cummins.
schematic representation of the linear cassette that was used is shown in Figure
Husnik et al. (2006). Evidence that the malolactic cassette was integrated
into the URA3 locus of the parental yeast S92 is provided in Figure 3 (Husnik
et al., 2006).
regulatory genes were present on the linear cassette as was stated by Cummins.
further stated that the plasmid bearing a selectable phleomycin marker gene
and frequently lost from the yeast cell”. Scientific evidence that the
plasmid, used only
for co-transformation and initial screening purposes, was indeed lost is provided
Figure 4 (Husnik et al., 2006).
In the latter part of paragraph 2 of his report, Cummins confuses the
construction of the malolactic yeast with the genetic construction
of the malo-ethanolic
yeast (Volschenk et al. 2001).
In paragraph 5 Cummins states, without providing
an explanation, “Numerous
translocations have been observed uniquely in wine yeasts and such chromosome
rearrangements involving transgenes can lead to unexpected toxicity in the
final product”. DNA cannot be toxic and Cummins provides no explanation
for this comment.
It is important to note that living cells, including wine yeasts, have mobile
elements called transposons (Lewin, 1990). These Ty elements insert themselves
different loci in the yeast genome at a frequency of 10-7 to 10-8. They can
or inversions that damages the chromosome and these recombination events in
yeasts may create novel open reading frames (ORF’s) that encode for proteins
not been characterized or it may disrupt ORF’s and prevent expression
of certain genes.
Recombination events in “natural” wine yeasts are thus ongoing
processes and it is
unpredictable in contrast with the integration event in ML01 that was targeted
characterized at the level of the genome, transcriptome, proteome and metabolome.
controlled and fully characterized recombination event in wine yeasts (such
as in ML01)
is unacceptable to wineries, regulatory bodies and the public, the use of all “natural” wine
yeasts with ongoing, uncontrolled and uncharacterized recombination events,
In paragraph 6 of his report, Cummins elaborates
on the presence of yeast nucleic acid and proteins in wine. He is apparently
also concerned about the presence of the malolactic enzyme and the malate
permease protein in wine produced with ML01. The argument that small amounts of DNA and proteins from ML01 may persist in
not relevant since no DNA or proteins foreign to the wine
making process were introduced into the malolactic wine yeast ML01. Wines produced by bacterial
malolactic fermentation may also contain small amounts of DNA including the
mleA gene encoding the malolactic enzyme; the protein could also be present. The
argument can be made for the malate permease genes from O.
oeni and S. pombe which
are present in wine. It should be noted that it is unlikely that the malate
(from O. oeni, S. pombe or ML01) that contains many transmembrane domains,
present in wine since this protein is hydrophobic and will not remain in solution.
In paragraph 7 Cummins states that the “genetic characteristics of the
the abandoned winery persisted for over ninety years”. Did yeast cells
survive that were
used 90 years ago or was DNA from these yeasts found? Were these yeasts fingerprinted
90 years ago to enable Cummins to come to the conclusion that it was the
The same arguments apply for yeast had been used in winemaking at least as
far back as
3150 BC. Yeast cells cannot survive that long without nutrients; we have
found that cells
survive for a maximum of three years in wine without any fresh nutrients
We have indeed considered that the ML01 yeast, same as other wine yeasts
parental strain S92, might become resident in a winery. It is for this very
reason that we
tested what number of ML01 yeast cells is required to conduct the malolactic
fermentation. From Figure 5 (Husnik et al., 2007) it is clear that no malolactic
fermentation occurred when less than 1% of ML01 yeast was present in the
inoculum at the start of fermentation. Even without washing and cleaning
the fermentation tanks in
which ML01 was previously used, it will be impossible to reach a population
cells/ml of ML01 under the worst circumstances. Apart from the malolactic
which is conducted by ML01 only when high cell numbers are present, ML01
to the parental strain S92 and resident ML01 cells in a winery is therefore
of no concern.
In a second and similar report on the internet (Institute of Science in
Cummins repeats his faulty statements previously
discussed in this document despite the fact that by now he had read our
paper in which
we described the genetic construction of ML01 (Husnik et al., 2006). In
claims that “ML01, was found to be only somewhat substantially equivalent
unmodified yeast, as a cytochrome p450 enzyme protein (?) appeared to have
altered from the parental strain based on a comprehensive analysis of yeast
and a number of codon changes were observed in the malolactic gene cassette,
changes were not considered significant”. Cummins clearly misinterpreted
our data as
far as the lanosterol 14-demythylase cytochrome P450 is concerned. Cellular
ML01 and S92 (parental strain) were identified and quantified by multidimensional
chromatography and tandem mass spectrometry. The concentration of only
lanosterol 14-demethylase cytochrome P450, was shown to be different at
a p-value <
0.05 across duplicate experiments. Lanosterol 14-demythylase cytochrome
P450 had a
weighted average ratio of 0.799 (using the S92 data as the denominator).
was not altered at all, the concentration of this enzyme in ML01 was slightly
(0.799) than what was found in the parental yeast. It is important to note
concentration of many proteins will differ among different wine yeast strains
differences in ploidy. All tests conducted showed that ML01 was substantially equivalent to the parental strain S92 (Husnik
et al., 2006) and not “somewhat” as
by Cummins. He also stated that number of codon changes
were observed in the
malolactic gene cassette, but those changes were not considered significant”. The PGK1 promoter, PGK1 terminator and partial URA3 flanking sequences do not code
and therefore don’t contain codons; only open reading frames coding for
have codons. There are two base pair changes present in the mleA gene in
compared to the sequence of one published mleA gene. Several mleA gene
now available (NCBI) and an alignment of all seven published sequences
reveals that the
“two unintended genetic changes” are in fact natural single nucleotide
the mleA gene. We have aligned the sequences of seven mleA genes from O.
genetic polymorphisms in question are highlighted in yellow. Other polymorphisms
the mleA gene in seven O. oeni strains are highlighted in red. Both glutamate
aspartate (nucleotide 1614) are thus present at this position in natural
strains of O. oeni used by the wine industry (see
reply to Biowatch SA).
2. Erica Martenson
Martenson discusses “The dangers of genetically modified wine yeast” on
Food Consumer and other web sites. http://foodconsumer.org/7777/8888/C_onsumer_A_ffair_26/The_dangers_of_genetically
In her second paragraph Martenson states “This yeast is available only
America where GMO’s are unregulated.” This statement is simply wrong.
The use of all
genetically improved cells in Canada is subject to approval by Health Canada
aspects) and Environment Canada (environmental safety). These two regulatory
are among the strictest regulatory bodies in the world. ML01 has been found
wine making and for release into the environment. Genetically engineered
strains of S.
cerevisiae have been exempted from EPA review in the USA due to the long
and safe use
of this yeast.
Her arguments why ML01 is “dangerous” is based on the faulty and
arguments of Cummins that have been addressed in this document. She clearly
read any of our scientific papers or Notifications on ML01. The Australian
that are opposed to the use of GMO’s until other countries use the ML01
that “What are the risks associated with using ML01? In terms of health
should be none. The two foreign genes incorporated into the wine yeast to
make it MLF-
competent come from organisms that are typically associated with foods and/or
beverages. One comes from the yeast Schizosaccharomyces pombe, which is found
many alcoholic beverages, and the other comes from O. oeni, which is used
the wine industry for MLF”. They further commented “It would seem
some of the more obvious risks and benefits associated with the use of ML01,
access to this yeast might be a good thing for Australian winemakers”. http://www2.awri.com.au/infoservice/media/releases/nogogmo.asp
malolactic yeast ML01 is as safe but better characterized than agricultural
crops obtained by traditional methods. Traditional plant improvement
relies on (1) mating
of two plants of the same species; (2) interspecies hybridization which
relies on the
transfer of “alien” genes between different but related species and
(3) wide crosses
between members of different genera. These wide crosses, which do not occur
introduce many entirely new genes into crop plants. For example, Triticale
artificial hybrid of such wide crosses between wheat and rye (Miller and
Furthermore, mutation of seeds or young plants by chemicals or radiation
often kills most
plants exposed to this treatment; some survive and are screened for required
traits. All of
these traditional treatments lead to genetic modification of the seed or
plant; the final
plant is not screened for unwanted mutations or recombination. The public
and many of
the self-appointed “experts” who oppose products of genetic engineering,
ignore the fact
that “plant breeding” produces uncharacterized genetic events which,
is far less accurate
and predictable than what can be achieved by recombinant methods in yeast.
In the end,
plant breeding and genetic engineering both entail genetic modification of
Clearly, it is not the process but rather the products that should be carefully
Martenson also tries to discredit ML01 by stating that “the pombe
yeast is found
in Africa and used to make beer”. The yeast Schizosaccharomyces
first isolated from beer in Africa but it has subsequently been isolated
from wineries and
it is now commercially available (as ProOenol) for deacidification of wine.
states that “a developer has an interest in getting its
product to market as soon as
possible, whether it has been proven safe or not”. It has taken us 14 years
to develop and
test the ML01 yeast before it was commercialized. One can hardly argue that
was brought to the market “as soon as possible”. No other
genetically modified cell,
obtained by traditional breeding or recombinant methods, has been characterized
same extent as the malolactic wine yeast ML01. Furthermore, independent
environmental studies conducted at the University of Stellenbosch have
shown that the ML01 strain
behaves in an identical fashion to the parental strain and it does
not affect growth or
survival of soil bacteria.
In her final paragraph Martenson states that “In our society, we often
our rights and discuss very little our responsibilities – to our
It is hardly
responsible to indoctrinate and confuse the public on the internet without
If she has the interest of the public at heart, she should also ask the
wineries that she has
listed if their wines are free of bioamines and ethyl carbamate, a carcinogen
found in food
and alcoholic beverages. We are poisoning our bodies with chemicals present
in food and
alcoholic beverages that we consume on a daily basis. This is contributing
to the fact that
healthcare has become unstainable in many countries. As a scientist I have
made it my
responsibility to use any safe technology, including genetic engineering,
to rid alcoholic
beverages and food of toxic compounds.
3. Objection to the use of ML01 in South Africa by Biowatch SA and
The objections of Biowatch SA to the use of ML01 in commercial wineries
South Africa and our response to the objection can be found in
the following document
4a. Objection to the use of ML01 in South Africa by The African Centre
The objections of The African Centre for Biosafety to the use of ML01
commercial wineries in South Africa is presented in the following
4b. Response to the Objections of The African Centre for Biosafety
Response to the Objections of The African Centre for Biosafety (PDF).
5. Literature cited
Lehtonen, P., 1996. Determination of amines and amino acids in wine: A review.
Enol. Vitic. 47, 127-133.
Lewin, B. 1990. Genes IV. Oxford University Press. pp. 680-682.
Lonvaud-Funel, A., 2001. Biogenic amines in wines: Role of lactic acid
Microbiol. Lett. 199, 9-13.
Medina, M.A., A.R. Quesada, I.N. de Castro and F. Sanchez-Jimenez. 1999.
Histamine, polyamines and cancer. Biochem. Pharmacol. 57, 1341-1344.
Miller, H.I. and G. Conko. 2004. The Frankenfood Myth. Preager, London.
pp. 1-269. Pryme, I.F. and S. Bardocz. 2001. Anti-cancer therapy: Diversion
of polyamines in the
gut. Eur. J. Gastroen. Hepat. 13, 1041-1046.
Santos, M.H.S. 1996. Biogenic amines: Their importance in foods. Int.
J. Food Microbiol. 29:213-231.
Soufleros, E., M.L. Barrios, and A. Bertrand. 1998. Correlation between
the content of
biogenic amines and other wine compounds. Am. J. Enol. Vitic. 49:266-278.
Volschenk, H., M. Viljoen-Bloom, R.E. Subden and H.J.J. van Vuuren. 2001.
Malo-ethanolic fermentation in grape musts by recombinant strains
cerevisiae. Yeast: 18:963-967.
Wallace, H.M. and R. Caslake. 2001. Polyamines and colon cancer. Eur.
Hepat. 13, 1033-1039.
Wantle, F., M. Gotz and R. Jarisch. 1994. The red wine provocation test:
histamine as a model for food intolerance. Allergy Proc. 15, 27-32.
Zee, J.A., R.E. Simard, L. Lheureux and J. Tremblay. 1983. Biogenic-amines
Am. J. Enol. Vitic. 34, 6-9.