Metabolic engineering of yeasts for high ethanol biofuel production may generate toxic metabolites and pose unique threats to agriculture. Prof. Joe Cummins
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 , I-SIS 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.
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).
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 .
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 .
Article first published 12/01/07
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