Complete sequences of pIJ101-based Streptomyces-Escherichia coli shuttle vectors.

Abstract

High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces-Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.