Shaping the Evolvability of Primordial Protein Maquettes by Indel Mutagenesis in Ultrahigh-Throughput Directed Evolution

BBSRC strategic theme: Transformative technologies

Directed protein evolution represents the current method-of-choice for enhancing select protein properties or imbuing it with novel functions, leveraging nature-inspired selection of improved variants from a large pool of diverse sequences. Traditionally, this diversity is achieved by rounds of substitution mutagenesis - probing which residues are adaptive in different positions within the protein. Substitutions represent the most common evolutionary events in natural evolution and can be conceptualised as small steps across the fitness landscape to fine-tune protein function. However, further mechanisms play a crucial role in natural protein evolution, such as small or large-scale insertions/deletions (InDels), gene duplications or recombinations, here termed exotic evolutionary events (EEEs). These are currently under-explored in directed evolution campaigns and their effect on protein evolvability compared to substitutions is largely unknown.

Firstly, evolution campaigns are often hindered by the ruggedness of the fitness landscape and the non-additivity of certain mutations, trapping proteins in low-fitness valleys and requiring iterative rounds of mutagenesis and screening. My project aims to explore whether EEEs may help in escaping these fitness valleys and thus enhance protein evolvability due to the large leaps through the landscape and the radical modification of the protein backbone.

Secondly, substitution libraries may hinder the rapid evolution of biocatalysts towards novel substrates or novel reactions, again due to the non-radical changes to their sequence. Previous work in the group suggested that while small-scale InDels are more often detrimental to protein fitness, they are more likely to yield an improved variant than substitution libraries only (Emond et al., 2020). Expanding on this, I aim to determine the effect of the EEEs on their ability to rapidly evolve a model protein system towards new substrate specificities and catalysed reactions.

Thirdly, previous work in the group demonstrated how large-scale protein truncations may benefit early enzyme evolution, bridging the barrier between small inactive proteins and functional de novo catalysts (Schnettler et al., 2023). Going further, EEEs may have played an even bigger role in the transition from small promiscuous biocatalysts to the large finely-tuned proteins of today, due to the resulting increase in mass and sequence complexity and therefore an increased ability to specialise in their function. Here, I aim to understand the role of EEEs in the early transition from small polypeptide biocatalysts to fine-tuned large proteins.

Until recently, probing the effect of these EEEs was exceedingly difficult due to their overall detrimental effect on protein fitness, requiring ultra-high-throughput screening technology to capture the rare improving variants. To overcome this barrier, I aim to use the existing ultra-high-throughput microfluidic screening technology (Colin, Zinchenko and Hollfelder, 2015), together with the methodology developed in the lab to generate high-quality protein libraries of amino acid InDels (Emond et al., 2020). As my model protein system, I will use the de novo evolved minimalist cAMP phosphodiesterase (Schnettler et al., 2023), allowing me to explore all three aspects of my project. Overall, this project aims to improve our understanding of various mechanistic features of enzyme evolution and the role the EEEs play in it.