Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Tolle and her co-workers for this research - Oehm, Hoesl, Treiber-Kleinke, Bozukova, Albers, Bukari, Semmler, Reppsilber, Ignatova, Gerstein, Budisa and our Key Account and Technology Officer Lauri Peil have published a research to test the limits of evolution and the potential for creating artificial diversity using top-down approaches to create synthetic life.

Synthetic parallelism

In this study, the researchers aimed to investigate the possibility of evolving an existing species, specifically the E. coli bacterium, to have an altered set of canonical amino acids.

The goal was to create synthetic life with an artificial diversity by inducing proteome-wide replacement of a canonical amino acid, in this case, tryptophan (Trp), with a non-canonical analogue called L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa).

Through this research, altering and challenging the genetic code, which has maintained its rigidity, allows for the exploration of unprecedented possibilities in the creation of artificial life.

Challenging the Genetic Code

Figure 1. Chemical structures of tryptophan and L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). The brackets indicate the structures of the precursor’s indole and β-thieno[3,2-b]pyrrole ([3,2]Tp). (Adapted from Tolle et al 2023)

It began with the question of whether replacing more than 20,000 Trp indole side chains in proteome with thienopyrroles would constitute important changes in the chemistry of life.

The goal of the experiment was to replace the tryptophan (Trp) in the genetic code with an analogue known as L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). This substitution meant that the Escherichia coli had to adapt to the environment using thienopyrrole instead of indole, which is usually used to induce a stress response. This approach consequently led to reassignment of UFF codons.

The genetic code of the E.coli was therefore altered and differed from its ancestor cells, signifying a transformation in the chemistry of life itself.

Figure 2. ALE on [3,2]Tp. Phase 1: the indole concentration was reduced from 1 µM to 0 µM in several steps. Phase 2 all other 19 cAAs were removed following the metabolic blocks pattern, and growth temperature was reduced to 30°C in order to remove the last amino acid, Met, from culture medium. The numbers in the bars above the graph denote the total number of the remaining cAAs. Phase 3 The temperature was gradually increased back to 37°C. (Adapted from Tolle et al 2023)

Reducing Amino Acids to Zero

In the second phase, the number of canonical amino acids was systematically reduced from 19 to 0.

Over generations, the evolving population accumulated mutation in genes associated with chemotaxis and flagella synthesis, common responses to stress in E.coli.

This adaptation process revealed a variety of results:

1) The ability of the E.coli to mitigate its stress response
2) Prioritise its cell growth in the face of dwindling resources
3) Stress-related proteins gradually decreased over time, returning to levels seen in the ancestral strain under non-stressful conditions

These findings were confirmed by genomic data, identifying mutations related to stress proteins and alteration in the master regulator RpoS.

Interestingly, the adapted strain seemed to counteract the negative effects of [3,2]Tpa incorporation by reducing its protein quality management, specifically by decreasing proteolytic activity.

Mutation in three proteases provided supporting evidence for this observation.

Mutations in Stress Response

In three different strains of bacteria isolated throughout the experiment (TUB85, TUB145, and TUB170), mutations were found in genes lon, ftsH, and clpP. These mutations were predicted to be harmful to the bacteria by a computer program called PROVEAN.

These mutations may cause problems in the way proteins fold because of the differences between [3,2]Tpa (a special type of amino acid) and Trp (a normal amino acid).

Figure 3. Mutations involved in the RpoS-mediated stress response. (A) Schematic representation of the mutations directly involved in the general stress response and their impact on the stress response. (Adapted from Tolle et al 2023)

The key findings of the study include the identification of mutations in genes associated with amino acid metabolism, such as astB, aroG, leuS, and rpoS. The mutations allowed the evolved strain to adapt to the absence of Trp and the presence of [3,2]Tpa.

Notably, the researchers observed changes in stress responses, chemotaxis, flagella synthesis, and proteolytic activity.

Creating Parallel Biological Worlds

The study demonstrated that adapting to proteome-wide non-canonical amino acid (ncAA) insertions could have extensive effects throughout the cell, affecting various cellular processes, and leading to a complex interplay of interactions.

The importance of this research lies in advancing synthetic biology through a top-down approach, where the genetic code of existing life forms is gradually changed to create synthetic cells with different genetic codes.

While various theories have attempted to explain the selection of amino acids in the genetic code, experiential verification remains essential.

The researchers suggest that the evolutionary alienation of the basic chemical makeup of living cells could very well be a promising approach to experimentally create parallel biological worlds with entirely distinct genetic codes.

For more detailed overview of the study, click here.