Multifunctional SEVA shuttle vectors for actinomycetes and Gram-negative bacteria

Actinomycetales, such as the genusStreptomyces, are well-known cell factories employed to produce a wide variety of secondary metabolites for industrial use. However, not only is the genetic engineering ofStreptomycesmore complicated and tedious than other model laboratory species, such asEscherichia coli,there is also a considerable lack of genetic tools, hindering its adoption as a common chassis for synthetic biology.In this work, 23 novel shuttle vectors are presented that follow the canonical SEVA (Standard European Vector Architecture) common architecture with the goal of increasing the genetic toolbox repertoire forStreptomycesand other actinomycetes. The ORI module of these plasmids is composed of the combination of two origins of replication, one for Gram-negative bacteria and the other forStreptomyces, a Gram-positive bacteria. Origins of replication have been included in the collection for integrative, low-copy number, and medium-to-high-copy number vectors forStreptomyces.Also, a new selection marker has been developed that confers resistance to apramycin. The functionality of these plasmids was tested via the heterologous expression of GFP and the heterologous production of the plant flavonoid apigenin inStreptomyces albusJ1074, with successful results in both cases, therefore expanding the current repertoire of genetic manipulation tools inStreptomycesspecies.