FireProtASR: Fully Automated Pipeline for Resurrecting Stable and Expressible Ancestral Proteins

Robust and stable enzymes accelerating chemical reactions with high specificity represent one of the keystones of metabolic engineering. During the course of evolution, nature produced a large number of diverse solutions to enzymatic catalysis by evolving natural proteins. While most of these ancestral proteins have been lost due to extinction, or to the march of evolution, it is possible to statistically infer their sequences using Ancestral Sequence Reconstruction (ASR)¹. ASR approach has been used to resurrect ancestral proteins, to identify key amino acid residues in metabolic enzyme complexes², to engineer thermostable enzymes³, to deduce information about evolutionary events and natural history⁴, as well as, to engineer enzymes with dual, catalytically distinct activities⁵. The latter is possible by reconstructing ancestors of enzymes that have catalytically differentiated, and evolved along separate trajectories.

ASR is generally not accessible to scientists outside communities of evolutionary biologists. The purpose of this research is to construct a fully automated pipeline that allows anyone, including those who lack specialist knowledge, to perform ASR, thus reducing the academic barrier to entry. The pipeline was verified against work that was previously done in the lab; on the ancestral sequence reconstruction of haloalkane dehalogenase family⁵. The pipeline is accessible online through the FireProt^ASR web server ⁶ at: https://loschmidt.chemi.muni.cz/fireprotasr/. The server can be used for designing stable, highly expressible and catalytically active enzymes for metabolic pathways, that can be used for the construction of cell factories producing high-value chemicals.

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