Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

Abstract

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. 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 refolding in vitro is very slow and its expression in E. coli leads 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 refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

“…Ancestral sequence reconstruction has been amply used in the post-genomic era as a tool to address fundamental problems in molecular evolution [3][4][5]. Furthermore, it has been found that ancestral proteins "resurrected" in the lab (i.e., the proteins encoded by the reconstructed sequences) often display interesting and even extreme properties [6][7][8][9][10][11][12][13][14][15][16] plausibly reflecting ancestral adaptations to unusual intra-and extra-cellular environments. Resurrected ancestral proteins often display high stability, supporting the frequently hypothesized thermophilic nature of ancient life.…”