Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic
engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To
further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients
via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal
serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1
or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and
Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination
based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces.