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

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, Em(r)), was constructed by ligation of pJY33 with pBluescript II SK(-) and an erythromycin resistance gene (Em(r)). 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.