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[SANET-MG] yeast modified for high ethanol production
ISIS Press Release 12/01/07
Yeasts Targeted for High Ethanol Production
Metabolic engineering of yeasts for high ethanol biofuel production may
generate toxic metabolites and pose unique threats to agriculture Prof.
A fully referenced version of this article is posted on ISIS members’
Yeast genetic manipulation more precise than crop plants, but..
Fermentation of plant materials to make ethanol has been greatly
promoted as a means of producing sustainable biofuel  ( Biofuels for
Oil Addicts , SiS 30) Production of ethanol during fermentation has been
limited by the inability of yeast to grow at high ethanol levels, and a
great deal of effort is being devoted to creating yeast strains that
tolerate high ethanol levels, so they can continue fermentation to
produce higher concentrations of alcohol. This has the major advantage
of saving on energy involved in distilling and refining the ethanol.
Yeast genetic manipulation is far more precise than can be achieved in
crop plants, and the genes in yeast have been precisely altered by
mutations. One method developed for making high ethanol yeast, for
example, involves site-specific mutagenesis (see below) of regulatory
proteins controlling a metabolic network for ethanol production, to make
the yeast tolerate high levels of ethanol and glucose .
Although genes can be altered precisely to avoid collateral genetic
damage, changing a metabolic network will still result in unexpected
metabolic effects. The reason is that because genes are connected in a
complex functional network, one gene cannot be altered without affecting
many others  ( Living with the Fluid Genome , ISIS Publications).
Twelve years ago, Japanese scientists 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 . As the yeast is not intended for making
very strong beer for consumption but ethanol biofuel, toxin production
may seem less of a problem, provided the yeast strain can be completely
contained, which is well nigh impossible.
GM yeast is likely to contaminate or cross with native yeasts. If
unexpected toxins are produced because of metabolic network alterations,
then we are in real trouble. Bakers yeast is not a pathogen, but may
become one as the result of contamination. And toxic ethanol yeast in
the human gut would be hardly desirable.
What if this yeast escapes into the general environment and contaminate
the soil as it might well do? Some years ago, a GM bacterium, Klebsialla
planticola , engineered to produce ethanol from wood wastes was found to
inhibit the growth of wheat plants in every microcosm tested [5, 6] (
Ethanol from Cellulose Biomass Not Sustainable nor Environmentally
Benign , SiS 30). A GM yeast engineered to produce high concentrations
of ethanol released into the soil may spell catastrophe for agriculture
and food production.
Manipulating metabolic networks is a brand new field and clearly here to
stay. Organic farmers and the organic industry will soon be faced with
difficult decisions about organic foods. Can genes and networks
manipulated by precisely engineered DNA changes be considered organic?
We will have to decide soon, before the bureaucrats decide for us.
Legitimate Recombination and Site Specific Mutagenesis
Site-specific mutagenesis has been used to make yeast produce and
tolerant high levels of ethanol [2, 7]. The process involves changing
specific DNA code words in a particular gene. In the case of the high
ethanol yeast, a commercial kit called the quick change site-specific
mutagenesis kit was used. Short DNA chains (oligonucleotides) with
desired mutations were first prepared by the experimenters or purchased
at the oligonucleotide store. The quick change kit provides the tools
for changing the wild type gene carried on a bacterial plasmid. The
mutant oligonucleotide is annealed to the wild type gene in the plasmid,
which is amplified to produce plasmids carrying the gene with the
specific mutations. Next, the mutant gene is inserted into the yeast at
a specific locus, a step that involves legitimate recombination.
Genes to be inserted by legitimate recombination need to be flanked by
short sequences of the gene at the insertion site. Homologous
recombination inserts the DNA and disrupts the target gene, allowing
rapid selection of cells with the inserted gene. In the case of the
regulatory protein gene targeted to the uracil locus, the transformed
yeast colonies would be identified using replica plating on growth
medium lacking uracil. The colonies with the disrupted gene do not grow
on the medium lacking uracil but leave a ghost like pattern on the agar.
These colonies are picked off the uracil-containing master plates, and
grown up for fuller testing.
The main concern about the manipulation of metabolic network regulators
is that the genes have multiple effects and changing the activity of one
gene inevitably alters that of many others. Consequently, it may lead to
production of unpredicted toxins (see above).
Conventional mutagenesis still effective
Ultraviolet light has been used for many years to generate mutant
strains for laboratory and commercial applications. This technique has
created strains of brewer's yeast that produce high levels of ethanol
for brewing and bioethanol production . ‘Petite' yeasts are
mitochondria-deficient strains that can be produced by either nuclear or
mitochondrial mutations. One nuclear petite increased ethanol production
when it was cultivated on starch .
GM yeasts to ferment cellulose for bioethanol
Amorphous cellulose was digested to produce ethanol by Saccharomyces
cervisiae modified with endogluconase genes from the fungus Trichoderma
and a wild yeast Saccharomycopsis . The two genes were inserted in the
uraFUR1 (Uracilphosphoribosyltransferase) gene . A number of genetic
modifications are designed to make Saccharomyces cervisiae metabolize
the sugar xylose found in lignocellulose in agricultural and forest
wastes. Xylitol dehydrogenase and xylitol reductase genes from the yeast
Pichia stipilis were integrated into the chromosomes of S. cervisiae .
Xylose was converted to ethanol in the modified strain [11-13]. Not much
thought seems to have been given to the consequences of releasing such
modified yeast to the environment. S. cervisiae has not been pathogenic
in plants or animals but modified yeast may create novel pathogens, or
simply prevent crop plants from growing (see above).
Mutations and genetic modifications of Saccharomces cervisiae are being
promoted to boost ethanol production from fermentation of crop and
forest waste products. However, critical evaluation of the human and
environmental consequences of releasing the novel organisms has not been
forthcoming. Furthermore, it has been shown recently that ethanol from
cellulose biomass is neither sustainable nor environmentally benign .
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