A de novo protein confers copper resistance in Escherichia coli

Abstract

To survive environmental challenges, biological systems rely on proteins that were selected by evolution to function in particular cellular and conditional settings. With the advent of protein design and synthetic biology, it is now possible to construct novel proteins that are not biased by eons of selection in natural hosts. The availability of these sequences prompts us to ask whether natural biological organisms can use na€ ıve-non-biological-proteins to enhance fitness in stressful environments. To address this question, we transformed a library of DNA sequences encoding~1.5 3 10 6 binary patterned de novo proteins into E. coli, and selected for sequences that enable growth in concentrations of copper that would otherwise be toxic. Several novel sequences were discovered, and one of them, called Construct K (ConK), was studied in detail. Cells expressing ConK accumulate approximately 50% less copper than control cells. The function of ConK does not involve an oxidase, nor does it require two of the best characterized copper efflux systems. However, the ability of ConK to rescue cells from toxic concentrations of copper does require an active proton motive force. Further selections for growth in higher concentrations of copper led to the laboratory evolution of variants of ConK with enhanced levels of activity in vivo. These studies demonstrate that novel proteins, unbiased by evolutionary history in the natural world, can enhance the fitness of biological systems.Synopsis: Living systems evolve to adapt to potentially lethal environmental changes. This normally involves repurposing existing genetic information (i.e. sequences that were selected by billions of years of evolution). Here we show that a completely de novo protein, not derived from nature, can enable E. coli cells to grow in otherwise toxic concentrations of copper, demonstrating that living systems also have the capacity to incorporate and protopurpose entirely novel genetic information.

A Strategy for Combinatorial Cavity Design in De Novo Proteins

“…For future goals in synthetic biology, the most significant findings showed that several of the binary patterned proteins provide life-sustaining functions in vivo. One protein enables E. coli to resist otherwise toxic concentrations of copper [16], while others rescue auxotrophic cells harboring deletions of conditionally essential genes [9,17,18]. Recently, we described a novel protein that supports cell growth by functioning as a bona fide enzyme, both in vitro and in vivo [19].…”

Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity

“…Protein expression is controlled by a T5 promoter and Lac operator and expression was induced with 100 μg/mL IPTG. The presence of a strong promotor on a high-copy plasmid leads to high levels of protein expression, as has been verified for numerous natural and designed sequences expressed in this system (Patrick et al 2007; Soo et al 2011; Fisher et al 2011; Hoegler and Hecht 2016). …”

“…In natural systems, this is a very rare event. However, recent work with large collections of non-natural de novo proteins facilitated our discovery of an entirely novel protein, ConK, with a new function that confers copper resistance in E. coli (Hoegler and Hecht 2016). …”

Hierarchical design of artificial proteins and complexes toward synthetic structural biology

“…Interestingly, further molecular evolution experiments demonstrated that a de novo protein Syn-IF, an original bifunctional generalist which can rescue two different auxotrophic strains, ΔilvA and Δfes, was evolved into two distinctive monofunctional specialist proteins with enhanced activity (Smith et al 2015). In addition, a de novo protein rescued E. coli from toxic levels of copper (Hoegler and Hecht 2016). Binary code strategy: a semirational approach for creating structural and functional de novo proteins.…”