Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression. Gámiz Arco, María Gloria Risso, Valeria Alejandra Gaucher, Eric A. Gavira, Jose A. Naganathan, Athi N. Ibarra Molero, Beatriz Sánchez Ruiz, José Manuel Ancestral sequence reconstruction Computational modelling of protein folding landscapes Heterologous protein expression Proteins from uncultured organisms Obligate symbionts This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.-R. and E.A.G.), National Science Foundation Award #2032315 (E.A.G.), National Institutes of Health Award #R01AR069137 (E.A.G.), Department of Defense MURI Award #W911NF-16-1-0372 (E.A.G.), Spanish Ministry of Science and Innovation/FEDER Funds Grants RTI-2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.) and the Science, Engineering and Research Board (SERB, India) Grant MTR/2019/000392 (A.N.N.). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and the staff at ID23-1 beamline for assistance during data collection. J.M.S.R. designed the research. G.G.-A. purified the modern/ancestral chimeras and the thioredoxin variants; she also performed and analysed the experiments aimed at determining their folding kinetics and biomolecular properties. V.A.R. performed experiments addressed at determining the efficiency of heterologous expression and provided essential input for the molecular interpretation of mutational effects on expression efficiency. E.A.G. provided essential input for the evolutionary interpretation of the data. J.A.G. determined the X-ray structure of the symbiont protein and provided essential input regarding its interpretation and implications. A.N.N. performed the computational simulations of the folding landscape for thioredoxins and provided essential input regarding their engineering implications. B.I.M. and J.M.S.-R. directed the project. J.M.S.-R. wrote the first draft of the manuscript to which V.A.R. J.A.G. A.N.N. and B.I.M. added crucial paragraphs and sections. All authors discussed the manuscript, suggested modifications and improvements, and contributed to the final version. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1–2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations. 2021-11-26T10:53:06Z 2021-11-26T10:53:06Z 2021-12-03 info:eu-repo/semantics/article G. Gamiz-Arco, V.A. Risso, E.A. Gaucher, et al. Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression. Journal of Molecular Biology 433 (2021) 167321. [https://doi.org/10.1016/j.jmb.2021.167321] http://hdl.handle.net/10481/71777 10.1016/j.jmb.2021.167321 eng http://creativecommons.org/licenses/by-nc-nd/3.0/es/ info:eu-repo/semantics/openAccess Atribución-NoComercial-SinDerivadas 3.0 España Academic Press