# A new-engineered integrative tool to target the terminal compartment of the Streptomyces chromosome

## TL;DR

This paper introduces a new genetic engineering tool for Streptomyces bacteria that targets the terminal part of the chromosome, enabling efficient integration of transgenes.

## Contribution

A new integrative tool based on the Samy phage that targets the terminal compartment of Streptomyces chromosomes is introduced.

## Key findings

- Samy integrase mediates specific integration in six Streptomyces strains from different clades.
- The Samy-attB site is conserved and located in the terminal compartment of most Streptomyces chromosomes.
- Heterologous expression of albonoursin gene clusters from Samy, PhiC31, and R4-attB sites yields equivalent production levels.

## Abstract

Phages are a valuable resource for the genetic engineering of Streptomyces antibiotic-producing bacteria. Indeed, a few integrative vectors based on phage integrase are available to insert transgenes at specific genomic loci. Chromosome conformation captures previously demonstrated that the Streptomyces linear chromosome is organized in two spatial compartments: the central compartment encompassing the most conserved and highly expressed genes in exponential phase, and the terminal compartments enriched in poorly conserved sequences including specialized metabolite biosynthetic gene clusters. This study introduces a new integrative tool based on a recently described phage, Samy, which specifically targets the terminal compartment of its native host chromosome. Samy is related to PhiC31 phage and, like the latter, encodes a serine integrase. Whereas PhiC31 targets a site generally located near the origin of replication, the Samy integration site is one of the farthest known attB sites from it. We demonstrated that the Samy integrase efficiently mediates the specific integration of a non-replicating plasmid in six Streptomyces strains from distinct clades. Bioinformatic analyses revealed that the Samy-attB site is rather conserved and located in the terminal compartment of most Streptomyces chromosomes. Finally, heterologous expression of the albonoursin biosynthetic gene cluster from the Samy-, PhiC31-, and R4-attB sites yields quantitatively equivalent levels of production, though qualitative differences were observed. Altogether, these results demonstrate that the att-int Samy system expands Streptomyces genetic engineering tools by enabling targeted integration in the terminal chromosomal compartment.

• Samy-based integrative vectors are new tools for engineering Streptomyces strains.

• They target the terminal compartment, farthest from the origin in most strains.

• They facilitate efficient heterologous production of the albonoursin antibiotic.

The online version contains supplementary material available at 10.1007/s00253-026-13707-2.