The Genetic Incompatibility of Metallo-?-Lactamases: Synthetic Biology Lessons from Directed Evolution

Synthetic biology is heavily reliant on the use of ‘standardized components’ with defined functions that can be recombined to create new systems. But can biological components really be standardized? Are genes interchangeable and modular? Numerous studies exploring heterologous protein expression have demonstrated that the answer is often no, but our understanding of the molecular mechanisms for such gene incompatibility remains poor. Here, we used detailed biochemical and biophysical characterization in combination with directed evolution to unveil the molecular mechanisms that constrain the heterologous expression of metallo-β-lactamases (MBL), a family of enzymes that confer antibiotic resistance to host bacteria.

The characterization of eight MBL genes in E. coli revealed that these enzymes provide drastically different levels of resistance to their host despite similar catalytic efficiency. The level of periplasmic expression for each gene was instead found to drive the level of functional resistance. To determine (1) if the host-specific processes that control periplasmic expression (transcription, translation, and translocation) present a barrier to the functional expression of a heterologous gene, and (2) how these barriers may be overcome, three MBL’s (NDM-1, VIM-2 and IMP-1) were evolved to confer higher resistance against ampicillin to E. coli. The level of resistance conferred by each gene was increased 64 to 128-fold over 18 rounds of directed evolution, yet the catalytic efficiency of each enzyme was largely unchanged. Mutations served to ‘domesticate’ each gene in the E. coli host by improving the efficiency of each of the underlying processes that contribute to periplasmic expression. This work has meaningful implications for the use of ‘standardized components’ in synthetic biology: Our results demonstrate that there are host-specific constraints on the functional expression of heterologous genes in foreign host organisms, but that these may be overcome with the use of directed evolution.