New Paper in ASM: De Novo Emergence of Peptides That Confer Antibiotic Resistance

About four months ago I came across an interesting PhD thesis on the evolution of antibiotic resistance (by Michael Knopp), and one of the papers published for the thesis work was described as being on the experimental de novo evolution of peptides that give antibiotic resistance. I couldn’t find the paper anywhere at the time so I wrote the author asking for a copy, and was informed it was still undergoing review so had yet to be published.

Knopp wrote back to me a few days ago and informed me the paper is now published. I haven’t finished reading it through but it already looks very interesting, and appears to be relevant to many of the discussions we’ve had around here on de novo evolution of ORFan/taxonomically restricted genes, including the ever relevant question of the density of function in protein sequence space.
Knopp M, Gudmundsdottir JS, Nilsson T, König F, Warsi O, Rajer F, Ädelroth P,
Andersson DI. De Novo Emergence of Peptides That Confer Antibiotic Resistance. MBio. 2019 Jun 4;10(3). pii: e00837-19. doi: 10.1128/mBio.00837-19.


The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes.


It says in results and discussion:

The diversity of each library was between 0.3 × 10^8 and 1.7 × 10^8, which is the typical upper limit of transformation efficiency. However, it is noteworthy that these libraries only cover a marginal fraction of the complete possible diversity, which is approximately 9.5 × 10^33 for a random 50-mer protein.

There’s a miscalculation in the calculation of the total sequence space of 50’mer proteins. It’s not ~9.5x10^33 as reported in the paper, it’s ~1.3x10^65 which is roughly 32 orders of magnitude larger.

Whoever did the calculation did 50^20, instead of 20^50.

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Better than one hit for kanamycin in a sample of only 10^8 random sequences. Just like catalytic antibodies!

I’d love to see @Agauger’s spin on this…


Did humans design the experiment and set up the conditions? Well there you go… QED: Design!



This is a pretty impressive piece of methodology!