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Abstract <p><i>Streptomyces</i> species are prolific producers of bioactive natural products, yet their genetic manipulation remains constrained by inefficient DNA delivery methods in many strains. Conjugation from methylation-deficient <i>Escherichia coli</i> has become the preferred approach for introducing plasmids into <i>Streptomyces</i>, relying on the presence of the <i>oriT</i> sequence within the mobilizable plasmid and the conjugation machinery (<i>tra</i> genes) encoded on the non-mobilizable helper plasmid pUZ8002. Among these, <i>traJ</i> encodes an essential component of the relaxosome. An additional copy of <i>traJ</i> is present downstream of <i>oriT</i> in some mobilizable plasmids, whereas many other commonly used plasmids lack <i>traJ</i>. Here, we investigated the impact of including <i>traJ</i> in mobilizable plasmids on conjugation efficiency by engineering two <i>oriT</i>-containing plasmids that initially lacked <i>traJ</i>: the ΦC31 integrative vector pRASK-SP44 and the non-replicative transposon delivery vector pHL734. We also examined the effect of introducing a second copy of <i>traJ</i> into the recombination-based chromosomal end-removal vector pCER. Incorporation of <i>traJ</i> into pRASK-SP44 and pHL734 resulted in tenfold and 100-fold increases in transconjugant numbers, respectively. Furthermore, introducing a second copy of <i>traJ</i> into pCER led to a fivefold improvement in plasmid transfer. Our data suggest that the inclusion of <i>traJ</i> improves transfer efficiency and may help overcome limiting steps in conjugation from <i>E. coli</i> to <i>Streptomyces</i>. Modulating the presence and copy number of <i>traJ</i> could represent a simple yet effective strategy to enhance genetic accessibility in <i>Streptomyces</i>. These findings have broad implications for the optimisation of genetic tools used in <i>Streptomyces</i> genome engineering and natural product discovery.</p> Key points <p>• <i>traj in mobilizable plasmids enhances conjugation to S. coelicolor.</i></p> <p>• <i>traj increases plasmid transfer efficiency up to 100-fold in S. coelicolor.</i></p> <p>• <i>traj may aid development of genetic tools for genome engineering in Streptomyces.</i></p>

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Enhancing conjugation from E. coli to Streptomyces coelicolor by incorporating traJ into mobilizable plasmids

  • 期刊论文

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  • 发表日期：

    2025年12月9日

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  • Paula Valdés-Chiara,
  • Yago Concha,
  • Sergio Alonso-Fernández,
  • Angel Manteca,
  • Gemma Fernández-García

摘要

Abstract

Streptomyces species are prolific producers of bioactive natural products, yet their genetic manipulation remains constrained by inefficient DNA delivery methods in many strains. Conjugation from methylation-deficient Escherichia coli has become the preferred approach for introducing plasmids into Streptomyces, relying on the presence of the oriT sequence within the mobilizable plasmid and the conjugation machinery (tra genes) encoded on the non-mobilizable helper plasmid pUZ8002. Among these, traJ encodes an essential component of the relaxosome. An additional copy of traJ is present downstream of oriT in some mobilizable plasmids, whereas many other commonly used plasmids lack traJ. Here, we investigated the impact of including traJ in mobilizable plasmids on conjugation efficiency by engineering two oriT-containing plasmids that initially lacked traJ: the ΦC31 integrative vector pRASK-SP44 and the non-replicative transposon delivery vector pHL734. We also examined the effect of introducing a second copy of traJ into the recombination-based chromosomal end-removal vector pCER. Incorporation of traJ into pRASK-SP44 and pHL734 resulted in tenfold and 100-fold increases in transconjugant numbers, respectively. Furthermore, introducing a second copy of traJ into pCER led to a fivefold improvement in plasmid transfer. Our data suggest that the inclusion of traJ improves transfer efficiency and may help overcome limiting steps in conjugation from E. coli to Streptomyces. Modulating the presence and copy number of traJ could represent a simple yet effective strategy to enhance genetic accessibility in Streptomyces. These findings have broad implications for the optimisation of genetic tools used in Streptomyces genome engineering and natural product discovery.

Key points

• traj in mobilizable plasmids enhances conjugation to S. coelicolor.

• traj increases plasmid transfer efficiency up to 100-fold in S. coelicolor.

• traj may aid development of genetic tools for genome engineering in Streptomyces.