Here's another paper at the intersection of biology and chemistry: a way to check the activity of a huge number of mutated esterase enzymes, all at the same time.
Protein engineering is a hot field, as well it should be, since enzymes do things in ways that we lowly organic chemists can only envy. Instead of crudely bashing and beating on the molecules out in solution, an enzyme grabs each of them, one at a time, and breaks just the bond it wants to, in the direction it wants to do it, and then does it again and again. If you're looking for molecular-scale nanotechnology, there it is, and it's been right in front of us the whole time.
Problem is, enzymes get that way through billions of years of evolution and selection, and those selection pressures don't necessarily have anything to do with the industrial reactions we're thinking of these days. And since we don't have a billion years to wait, we have to speed things up. Thus the work at places like Codexis on engineered mutant enzymes, and thus a number of very interesting takes on directed evolution. (Well, interesting to me, at any rate - I have a pronounced weakness for this sort of thing).
This latest paper, from the University of Greifswald in Germany, builds on the work of Manfred Reetz at the Max-Planck Institute, who's been very influential in the field. Specifically, it follows up on the idea in this paper from his group and this one from the Quax group at Groningen in the Netherlands. That technique involved selecting for specificity in esterase enzymes by giving organisms a choice of two substrates: if they hydrolyze the right chiral starting material, the cleaved ester furnishes them with a nutrient. If they hydrolyze the wrong one, though, they produce a poison. Rather direct, but with bacteria there's no other way to get their attention - survival's really all they care much about.
And that technique worked, but it was a bit laborious. The largest number of different variations tested was about 2500, which seems like a lot until you do the math on protein mutations. It gets out of control very, very quickly when you have twenty variations per amino acid residue. Naturally, some of the residues shouldn't ever be touched, while others will have only minimal effects, and others are the hot spots you should be concentrating on. But which ones are which? And since you absolutely can't assume that they're all acting independently of each other, you have your work cut out for you. (Navigation through this thicket is what Codexis is selling, actually).
This latest paper adds flow cytometry, cell sorting, to the mix. Using dye systems and one of these machines to distinguish viable bacteria from dead or dying ones lets you take a culture and pull out only the survivors. When the authors expressed different esterases (with known preferences for the two substrates) in E. coli, they got the expected results - the ones with an enzyme that could cleave the nutrient-giving substrate grew, while the ones that unveiled the poison (2,3-dibromopropanol) halted in their tracks.
They then took another esterase with very modest selectivity and created a library of mutant variations - about ten million mutant variations - and expressed the whole shebang in a single liquid colony of E. coli. This was then exposed to the mixture of substrates, and anything that grew was pulled out by the cell sorter and plated out on agar (also containing the selection mixture of substrates). They got 28 clones to grow in the end, and characterized three of these more fully as purified enzymes. Of those, two of them were, in fact, much more selective than the starting enzyme (giving E values, enantiomeric ratios, of 80 to 100 as opposed to 3). Another, interestingly, was not selective at all.
And when you look at the sequences and the mutations that were picked out, you can see how tricky a business this is. One of the two selective enzymes had its valine-121 residues mutated to isoleucine (V128I) its phenylalanine-198 residue mutated to cysteine (F198C). The other was broadly similar, with one added mutation: that valine-121 was changed in this case to serine (V121S), the F198 was mutated to glycine (F198G), and also valine-225 was changed to alanine (V225A). Now, some of those aren't very big mutations (V to I, V to A), but what's even more interesting is the sequence of the unselective clone that they characterized: that one had V121I, F198G, V225A. So it had a mix of the exact mutations found in the two selective enzymes, but was itself a dud.
I'm glad to see that this worked, although you have to wonder how efficiently it moves in on a target when you get two decent hits out of ten million starting mutations. (The relative ease of screening goes a long way towards making up for that). But what I'd like to see is a mix of this technique with the one that I wrote about a few weeks ago, where a bacterium was evolved to use a chlorinated DNA base. That one used a particularly slick directed-evolution device, which would be quite interesting to apply to this food-versus-poison idea. You'd have to do some fine-tuning, especially at first, since the liberated poisonous substrate would be killing off the just and the unjust alike (which is the same problem that this current paper faced). But it seems like there should be a way to run things so that you're not just screening a big library of random mutations in the enzyme, but actually pushing the enzyme to evolve in the direction you want. Thoughts?