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Re: [SANET-MG] high ethanol bakers yeast



Hi Douglas and all,
I have tried to explain about yeast genetic manpulation in reply to Douglas.

*Legitimate Recombination and Site Specific Mutagenesis*

* Site specific mutagenesis: *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. Unfortunately the kit is not yet available at Wal-Mart. 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 born on a bacterial plasmid.. The mutant oligonucleotide is annealed to the wild type plasmid and the quickchange DNA amplifies the annealed plasmid to to produce plasmids with specifc mutations specifying altered amino acids in the protein regulating the ethanol network in yeast.Next, the mutant gene must be inserted into the yeast at a specific locus. That step invovles legitimate recombination.

*Legitimate Recombination:* Genetic engineering bacteria and yeast (and other fungi) is achieved using legitimate (homologous) recombination. Genes to be inserted require require short tails from the gene of the insertion site to target the gene insertion into a chromosomal locus. The inserted gene disrupts the target gene allowing rapid selection the inserted gene. In the case of the yeast ethanol control gene targeted to the uracil locus the transformed yeast colonies would be identified using replica plating on uracil lacking media. The colonies bearing the disrupted gene do not grow on the uracil minus media but leave a ghost like pattern on the agar. The colonies modified with the regulator gene are picked off the uracil master plates and grown up for fuller testing.

Crop biotechnology does not use legitimate recombination but instead requires illegitimate recombination in which the gene to be inserted slides into a double strand break in the DNA. The crop genetic modifications truly produce bastards which are the products of illegitimate recombination. Animal genetic engineering employs both legitimate recombination and illegitimate recombination. Illegitimate recombination tends to make the chromosomes of the recombinant crop unstable in part because of the needed array of control and selection genes needed to run the desired gene insert. Legitimate recombination is normally stable but when genes homologous to the inserted gene are already present at another locus then mitotic or meiotic recombination between the two will lead to translocations, duplications or deletions.

The main concern about the manipulation of network regulators is that the genes have highly pleiotropic effects. Consequently they may lead to production of unpredicted toxins. Such side effects must be looked for. On the up side bakers yeast was never a pathogen to anything , unless, of course, it was modified with a gene for a toxin.

The genome of baker’s yeast is fully sequenced and its genetic behavior has yielded a grand crop of PhDs and even Nobel prizes rivaling that of bread mold.

I hope that this discussion has helped to clarify the matters and serve as an introduction to the looming threat of network regulators.**



Douglas Hinds wrote:
Hi Joe and other sanet subscribers following this important thread:

Prof. Cummins wrote:

    Thank you for the interesting questions. I read the paper in
    science along with its accompanying on-line 32 page description of
    the genetic methodology used to create the high ethanol yeast.

    Before answering your questions I will go into the methodology a
    bit. I will mention that I taught Microbial Genetics for over
    twenty five years and published in both yeast and fungal genetics
    and molecular biology.

    The system developed for making high ethanol yeast is certainly
    genetic engineering but it is not genetic modification with
    transgenes.

    Key regulators for production of high level ethanol in the
    presence of high level glucose were identified in yeast. Those
    genes DNA sequences were altered by a process called site specific
    mutagenisis then reinserted into the yeast at a particular locus
    (the uracil gene).

Could you describe the process of "site specific mutagenisis" used
to alter the DNA sequences of the high level ethanol production
genes; and explain how researchers were able to reinsert these DNA
sequences into the yeast genome at a particular locus (the uracil
gene) with such great precision?

The fact that they were able to do this seems strange to me, because
as I understand it, when the GE organisms sold by Chem/Gene/Seed
companies like Monsanto and DuPont (which apparently participated in
the creation of the GE yeast referred to), were created, there was
no way to determine where within the genome the inserted gene would
end up, thus shifting the rest of the genome's components in the
process and as a result, making the genome itself unstable. Is that
correct, Professor? And if so, how is the process used by the MIT
group different? How were they able to take out specific genes and
reinsert them into the exact same place?

One significant difference may be the fact that no new genes are
inserted in this process, I suppose. Another significant difference
may be the fact that yeast is a much simpler organism (which
therefore, has a much simpler genome) than the genomes of the plants
cultivated by man. Likewise, more is known about yeast genetics than
is the case with corn, soy, cotton, rice and potatoes etc. Is that
correct?

    The regulatory genes controlled a number of genes in a metabolic
    network for ethanol production and most importantly, [the]
    ability to tolerate high levels of ethanol and glucose.

    The current work on such key regulators follows work on E coli
    ethanol production by modifying a sigma factor regulator of
    transcription. Douglas asked "However, we could call the effect I
    refer to "genome shifting", which adds an unknown, unforeseeable
    and uncontrollable element to the expected results. Do you agree?"
    I do not entirely agree.

    Yeast genetics are far more precise than the crude methods used in
    crop plants and the genes in the yeast have been precisely altered
    by mutations on knowingly altered DNA. Manipulating a metabolic
    network is new and breathe taking but could lead to unexpected
    toxins being produced.

    These will have to be checked on in the near future but since the
    yeast is not for very strong beer but for commercial ethanol fuel
    the toxins could be ignored. A former student and colleague, Inge
    Russel, developed a very high ethanol yeast by breeding and
    selection alone and her strain is presently better than the
    promoted higher tech system because it was bred from a commercial
    ethanol yeast rather than into the laboratory strains used in the
    high tech version. Lab yeast tend not to do all that well in
    commercial operations for brewing, wine making or ethanol making.

    The gene regulators are somewhat similar to the MADS box
    regulators growing popular in crop genetic manipulation that I
    discussed previously. However, the MADS box alterations have been
    transgenic while the network ethanol yeast are not.

    Douglas asks "To what extent is the GE yeast likely to cross with
    native yeasts existing in nature? Is the threat similar to that
    created by GE crops, or are GE yeasts likely to be less
    threatening? You ask an important question Douglas. My feeling
    this that regulators will have a hard time getting their teeth
    into genes that are precise;y altered within a particular
    organism.

    If unexpected toxins are produced because of metabolic network
    alterations then we are in real trouble. Bakers yeast is not a
    pathogen to anything , I believe, but it has been used as a
    probiotic. Killer beer is every boy's nightmare! However, the
    toxins should show up early and be dealt with promptly or even
    ignored for making fuel. However, toxic ethanol yeast in the gut
    would not be any fun at all.

    Manipulating metabolic networks is brand new but clearly here to
    stay. Organic farmers and the industry will soon be faced with
    difficult decisions about organic foods. Can genes and networks
    manipulated by engineered DNA code word changes within a crop be
    considered organic? We will soon have to decide.Let us hope the
    decision is ours and not the bureaucrats.

    Finally, many recollect that I objected to GM wine and I still do.
    That stuff is transgenic and should be labeled, dammit all. You
    cannot imagine the abuse I get from the developers of GM wine!

    Thanks so much for the important questions, excuse my rambling
    answers.

Your answers were clear and to the point, Professor. And we are very
fortunate to be able to be able to draw on an information source
coming from the highest possible level, both in terms of your
technical competence and scientific credibility but also (and
very significantly), in relation to the critical social and
environmental factors that come into play in the context of this
issue.

And for that all of us here thank you, Joe.

Douglas

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