Researchers try to cut the genetic code from 20 to 19 amino acids - Ars Technica

The genetic code is central to life. With minor variations, everything uses the same sets of three DNA bases to encode the same 20 amino acids. We have discovered no major exceptions to this, leading researchers to conclude that this code probably dated back to the last common ancestor of all life on Earth. But there has been a lot of informed speculation about how that genetic code initially evolved.

Most hypotheses suggest that earlier forms of life had partial genetic codes and used fewer than 20 amino acids. To test these hypotheses, a team from Columbia and Harvard decided to see if they could get rid of one of the 20 currently in use. And, as a first attempt, they engineered a portion of the ribosome that worked without using an otherwise essential amino acid: isoleucine.

First off, why would you do this? Most work in the field has focused on altering the genetic code in ways that are useful, such as using more than 20 amino acids to enable interesting chemistry.

The reasoning here seems to be that, prior to the last common ancestor of life on Earth, organisms experimented with various genetic codes and probably used a mix of proteins and catalytic RNAs to run their metabolisms. While we’ve done a lot of studies on catalytic RNAs, we have far less of an idea of what sort of chemistry is possible with a reduced genetic code. And the researchers suggest that AI-based tools have matured enough that redesigning proteins to use fewer amino acids is far more realistic than it was just a few years ago.

Isoleucine is one of three highly similar amino acids, along with leucine and valine. In the portion of the structure that’s distinct from other amino acids, all three have a branched structure that’s composed entirely of carbon and hydrogen. That makes them all hydrophobic, and they often are located in the interior of proteins, which keeps them away from the watery environment of the cell. So, purely by reasoning it out, one of those three would seem to be a good candidate to get rid of.

The researchers involved backed that reasoning up with evidence. They ran an analysis of the E. coli genome, checking which amino acids were substituted by other ones in related proteins from other species. Isoleucine was the amino acid that was most frequently swapped out for a different one. So, the researchers decided to start answering the question of whether we really need it at all.

Editing all 4,500 or so genes in E. coli would be a monumental task, and that many changes at once would almost certainly end up killing it, so the researchers started out with much smaller tests. To begin with, they took a set of 36 essential genes and replaced every isoleucine in them with valine, a similar amino acid, and then put the introduced gene back into the genome. For 22 of the genes, doing so killed the cells. But that does indicate that 17 of them got by ok without isoleucine, including one where it was swapped out in 45 different positions along the amino acid chain.

Notably, even in cases where cells tolerated the change, their growth often slowed compared to the unedited cells. That will become a recurring theme.

Redesigning the ribosome

To give their project a focus, the researchers decided to start engineering an isoleucine-free ribosome. The ribosome is a large complex of proteins and RNAs that translates messenger RNAs into proteins—you can think of it as a bit like one of the hardware components that’s needed to boot a living cell from a genome. Obviously, many of the proteins in the ribosome have critical enzymatic activities. But bringing that complex together requires that these proteins interact with each other and RNAs. So, the ribosome provides a stringent test of whether engineering out an amino acid can be tolerated by cells.

As a preliminary test, the team did an isoleucine-to-valine swap for 50 different individual genes that contribute proteins to the ribosome. Eighteen of those worked with no obvious problems, another 19 grew more slowly, and the changes were lethal for the remaining 13 genes. The team then focused on the 32 genes with reduced fitness and adapted deep-learning protein-design software to suggest alternative sequences that did not include isoleucine.

Iterative testing using four different software packages produced alternative protein sequences for 25 of these 32 proteins that eliminated the fitness issues.