Investigating Epigenetics as a Mechanism to Regulate Bacterial Heterologous Expression

Epigenetic DNA methylation in bacteria is involved in phage defense, and it can also affect gene transcription. Epigenetic methylation as part of restriction-modification systems is frequently considered a barrier in synthetic biology since it can prevent the introduction of heterologous genes to new host organisms. Due to this, many studies that construct bacterial hosts for use in synthetic biology often remove these restriction-modification systems. There are no studies however that have attempted to optimize restriction-modification systems and orphan DNA methyltransferases to boost heterologous gene product yields. For this study, our ultimate goal was to investigate whether epigenetic DNA methylation can be used as a tool to increase heterologous expression yields in bacteria. We carried this study out by obtaining and comparing DNA methyltransferase deletion mutants in a variety of bacterial species that have been widely used as heterologous hosts, namely gram-positive Bacillus subtilis, and gram-negative Escherichia coli and Pseudomonas putida. For the gram-negative strains, we confirmed the expected epigenetic methylation in the wildtype strains. Additionally for Pseudomonas putida, we generated four double-crossover deletion mutants and two single-crossover mutants. For B. subtilis we obtained deletion mutants from the Bacillus Genetic Stock Center. For each of two B. subtilis DNA methyltransferase deletion mutants, we compared it to the wildtype strain, B. subtilis 168, for differences in cell growth, motility, methylome, and metabolome. We found that epigenetic methylation in B. subtilis regulates the metabolome and serves as rationale for continuing the investigation on whether epigenetic methylation can be used to optimize heterologous gene expression.