Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33

•A cryptic plasmid, pJY33 (2365 bp), was isolated from Weissella cibaria 33.•pJY33 was used for the construction of a LAB-Escherichia coli shuttle vector, pJY33E.•pJY33E replicates in Lactococcus, Lactobacillus, Leuconostoc, and Weissella.•pJY33 replicates via RCR and aga gene was expressed by using pJY33E.

A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Emr), was constructed by ligation of pJY33 with pBluescript II SK(−) and an erythromycin resistance gene (Emr). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose.