David A. Elsemore asked:
>I have recently looked for E. coli genes that when overexpressed provide resistance of E. coli to the herbicide glyphosate. Glyphosate targets the enolpyruvylshikimate-3-phosphate synthase. One clone that allowed E. coli to grow in the prescence of glyphosate contained the glucokinase locus. My idea of a possible mechanism is that overexpression of the glucokinase is diverting pools of free glucose (no idea how much free glucose is in the cells, although some of the literature indicates pools are larger than previously suspected) into glycolysis and/or the pentose phosphate pathway and thereby increasing carbon flux through the biosynthetic pathway for aromatic amino acids. Is this completely crazy?
Glyphosate acts by inhibiting enolpyruvylshikimate-3-phosphate synthase
uncompetitively. The immediate effect is to increase the concentration
of its substrate, 3-phosphoshikimate, which has the secondary effect of
increasing the inhibition still further. As a consequence the reaction
that makes 3-phosphoshikimate is forced much closer to equilibrium than
it would normally be, and what you observe in plants that have been
killed with glyphosate is a huge (several hundredfold higher than
normal) concentration of shikimate. It is almost certainly this
accumulation of shikimate that kills the plant.
So, what does all this imply about the question? You cannot overcome
this kind of toxicity by pushing more carbon flux through the pathway:
this usually doesn't work anyway (because pathways are designed by
evolution to resist efforts to push more material through -- that's why
naive biotechnology doesn't work); but in this case even if it did work
it would make matters worse, because the concentration of shikimate
would certainly increase.
The alternative would be to pull more material through. This wouldn't
make the toxicity worse, but I find it difficult to visualize how it
could be achieved or that it would do anything positive either. I
suspect that if you found a way of removing
enolpyruvylshikimate-3-phosphate faster it wouldn't have any observable
effect on the inhibition, and hence on the toxicity.
Thus my guess is that the glucokinase effect you observe is a red
herring.
Incidentally, what do you want glyphosate-resistant strains of E. coli
for?
Athel
