Harnessing phage defense systems to develop a Burkholderia bacterial host

Heterologous production of metabolites and proteins requires the transfer of foreign DNA into a host. One common barrier to the genetic engineering of bacteria is their plethora of phage defense systems. Targeted deletion of defense system components can improve DNA transfer for downstream applications. We sought to improve the transformation efficiency of Burkholderia sp. FERM BP-3421, a β-proteobacterial host that has great potential for the high-titer production of bioactive small molecules. We investigated restriction-modification systems and the recently described Kiwa phage defense system. Through deletion of three restriction enzyme genes, transformation efficiency was improved nearly 7-fold. The electroporation protocol was also optimized with respect to temperature, cell density, and buffer composition, which resulted in an additional 40-fold improvement in transformation efficiency, reaching 1 × 10 ⁶ CFU/µg plasmid DNA. Interestingly, a mimicry by methylation approach, where DNA is pre-methylated using two host DNA methyltransferases, outperformed the transformation efficiency obtained with restriction enzyme deletion mutants nearly 3-fold. Moreover, contrary to previous reports in Escherichia coli , deletion of the Kiwa system did not improve transformation efficiency into Burkholderia sp. FERM BP-3421. Understanding the impact of bacterial innate immune systems on genetic tractability is vital to improving non-model host strains to produce high-value molecules of interest.

IMPORTANCE

The ability to transfer DNA into bacteria is key to genetic engineering. However, bacteria have evolved immune defenses to recognize and protect themselves from foreign DNA, thwarting genetic manipulation. Here, we identified putative defense systems in a β-proteobacterial strain of interest and characterized their impact on DNA transfer. The defense systems studied belong to the well-known class of restriction-modification systems and to the recently described Kiwa system. Intriguingly, we observed higher DNA transfer efficiency by avoiding restriction-modification systems via pre-methylation rather than by restriction gene deletion. Future studies should investigate the underlying mechanism for this difference. Moreover, contrary to what was reported for γ-proteobacteria Escherichia coli , we showed that the Kiwa system does not impair DNA transfer into the β-proteobacteria Burkholderia sp. strain. There has been significant interest in the discovery of novel defense systems. Learning more about their complexity is important to enable genetic manipulation of previously intractable strains.