Construction Of Plasmid Expression System In Lactobacillus Plantarum

Lactic acid bacteria(LAB)are the components of human intestinal microorganism.They are generally recognized as safe(GRAS)strains and widely applied in food industry,which afford advantages in expression system as the host bacteria more than other microorganisms.Here we isolated a Lactobacillus from the Chinese traditional food Lajiaoza.16 S rRNA and physiological analysis revealed it is Lactobacillus pentosus and given the name Lactobacillus pentosus JS1 A cryptic plasmid was isolated from L.pentosus JS1.Full length sequence of the plasmid was sequenced by primer walking method.The plasmid was13205 bp,after BLAST with NCBI database,the new plasmid was named pJS1..The G+C content of pJS1 was 35% which has 14 ORFs(open reading frame).Two replicons were identified by sequence analysis which were named ori1 and ori2,however,these two replicon has 80% nucleonic similarity.Both replicon havea specific 22 bp direct repeats region,ori1 has 4.8 repeats and ori2 has 4.5 repeats.ORFs of replication protein repB was adjacent to the repeats.To determine minimal replicon,different length fragments contains core repeats and ORFs were cloned into pUCM to construct Escherichia coli-lactobacillus shuttle vectors.The results showed that both ori1 and ori2 are workable in several strains of Lactobacillus.The minimal replicon included complete direct repeats sequence and repB,the putative replication initiation gene.The results of host range showed that the replicon could survive in L.plantarum and L.paracaseiwith a narrow host range.To determine the copy number of pJS1 in different growth phase of L.pentosus JS1,quantitative PCR results showed that the copy number of pJS1 was highest in the early logarithmic growth phase,which was 19 copies.However,the copy number gradually decreased to 7 copies in the stationary phase.pJS1 was a low copy plasmid.Continuous inoculation experiments in antibiotics free medium showed that the constructed shuttle vector based on ORI2 replicon was unstable in the host.The loss rates between generations was lass than 5%.we constructed a expression vector based on shuttle vector.The enhanced green fluorescent protein(eGFP)was used for protein expression.The protein of eGFP was successfully expressed in L.plantarum ST-III,L.plantarum LPHS and L.paracasei LPC-37.