Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses

We present a selection design that couples S-adenosylmethionine-dependent methylation to growth. We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution. We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone. Implementation of this design in Saccharomyces cerevisiae is also demonstrated. Author summary Many important biological processes require methylation, e.g., DNA methylation and synthesis of flavoring compounds, neurotransmitters, and antibiotics. Most methylation reactions in cells are catalyzed by S-adenosylmethionine (SAM)-dependent methyltransferases (Mtases) using SAM as a methyl donor. Thus, SAM-dependent Mtases have become an important enzyme category of biotechnological interests and as healthcare targets. However, functional implementation and engineering of SAM-dependent Mtases remains difficult and is neither cost effective nor high throughput. Here, we are able to address these challenges by establishing a synthetic biology approach, which links Mtase activity to cell growth such that higher Mtase activity ultimately leads to faster cell growth. We show that better-performing variants of the examined Mtases can be readily obtained by growth selection after repetitive cell passages. We also demonstrate the usefulness of our approach for discovery of Mtase-specific drug candidates. We further show our approach is not only applicable in bacteria, exemplified by Escherichia coli, but also in eurkaryotic organisms such as budding yeast Saccharomyces cerevisiae.