Biology subjects

Pisera, A.

Publications and source records attributed to Pisera, A..

3 recordsLinked to original sources

bioRxiv2025

Rapid continuous evolution of gene libraries towards arbitrary functions

Life relies on constant biomolecular innovation, but the slowness of natural evolution limits our ability to witness new gene functions emerging in real time. Consequently, much remains unknown about how - and how easily - functional biomolecules originate and evolve. To address this gap, we encoded diverse gene libraries (e.g., open reading frames from Saccharomyces cerevisiae and Escherichia coli) onto an orthogonal DNA replication (OrthoRep) system and exposed the resulting continuously hypermutating genetic repertoires to a multitude of complex selection pressures in vivo. This experimental template mimics the "multi-gene, multi-objective" possibility landscape of natural evolution, but at drastically accelerated evolutionary speed. In only [~]100 generations ([~]1 month), populations evolved an abundance of novel gene functions when challenged with diverse pressures including vitamin deficiency, metal toxicity, promoter inactivity, and protein degradation. Notably, several evolved outcomes originated from sequences with no initial effect on fitness, demonstrating both emergence and evolution of biomolecular function. Evolved outcomes proved highly potent, often increasing fitness by several-fold under selection. Evolved genes spanned a range of molecular functions including de novo metal binding, transcription factor activity, and modulation of protein degradation, as supported by biochemical and RNA-seq experiments. Overall, our findings show that novel gene functions can originate with surprising ease in cellular contexts and motivate further continuous evolution experiments that agnostically explore the fertility of sequence space in vivo.

evolutionary biology↗

bioRxiv2024

A System to Explore the Adaptive Dynamics of Multicopy Plasmids: The Role of Copy Number and Mutation Rate in Evolutionary Outcomes

Multicopy plasmids are widespread in nature and compose a common strategy for spreading beneficial traits across microbes. However, the role of plasmids in supporting the evolution of encoded genes remains underexplored due to challenges in experimentally manipulating key parameters such as plasmid copy number and mutation rate. In this work, we developed a strategy for controlling copy number in the plasmid-based continuous evolution system, OrthoRep, and used our resulting capabilities to investigate the evolution of a conditionally essential gene under varying CN and mutation rate conditions. Our results show that low CN facilitates the faster enrichment of beneficial alleles while high CN promotes robustness through the maintenance of allelic diversity. High CN also slows the removal of deleterious mutations and increased the fraction of non-functional alleles that could hitchhike during evolution. This study highlights the nuanced relationships between plasmid CN, mutation rate, and evolutionary outcomes, providing insights into the adaptive dynamics of genes encoded on multicopy plasmids and nominating OrthoRep as a versatile tool for studying plasmid evolution.

evolutionary biology↗

bioRxiv2024

Ultra-Efficient Integration of Gene Libraries onto Yeast Cytosolic Plasmids

Efficient methods for diversifying genes of interest (GOIs) are essential in protein engineering. For example, OrthoRep, a yeast-based orthogonal DNA replication system that achieves the rapid in vivo diversification of GOIs encoded on a cytosolic plasmid (p1), has been successfully used to drive numerous protein engineering campaigns. However, OrthoRep-based GOI evolution has almost always started from single GOI sequences, limiting the number of locations on a fitness landscape from where evolutionary search begins. Here, we present a simple approach for the high-efficiency integration of GOI libraries onto OrthoRep. By leveraging integrases, we demonstrate recombination of donor DNA onto the cytosolic p1 plasmid at exceptionally high transformation efficiencies, even surpassing the transformation efficiency of standard circular plasmids into yeast. We demonstrate our methods utility through the straightforward construction of mock nanobody libraries encoded on OrthoRep, from which rare binders were reliably enriched. Overall, integrase-assisted manipulation of yeast cytosolic plasmids should enhance the versatility of OrthoRep in continuous evolution experiments and support the routine construction of large GOI libraries in yeast in general.

synthetic biology↗