Construction Of The Food-grade Inducible Gene Expression Systems In Lactococcus Lactis And The Expression Of Fusion OprF/H From Pseudomonas Aeruginosa

Lactic acid bacteria (LAB) are GRAS bacteria that are widely used in food industry and medicine. It is important to establish food-grade expression vectors systems for production of functional proteins. Food-grade means the vector bacteria and the selectable marker should be safe enough to meet the needs of food production.The nisin-controlled expression (NICE) system of L. lactis is one of the most widely used expression systems of Gram positive bacteria. The NICE system for controlled heterologous and homologous gene expression in L. lactis has been proven to be very valuable.In the production of protein with cells, the secretion of heterologous proteins are an efficient way to escape intracellular proteolysis, and the replacement of SP_Nuc by the homologous SP_Usp45 and a fusion with SP_Usp45, Nuc, and LEISSTCDA can enhance the production yields and the secretion efficiency of heterologous proteins in LAB. The construction of food-grade inducible gene expression system and engineering strains will intergate the biological function of beneficial bacteria with the activity of expressed products of heterologous genes, and will have great potential in food and medicine.In the present study, we constructed a series of food-grade vectors harboring food-grade selectable marker a-aga, theta replicon, P_nisA -MCS-T_pepN and P_nisA-SP_Usp45-nucA-CWA_M6-tlt2 that were suitable for the production of cytoplasmic and secreted heterologous proteins in L. lactis. The vectors are controlled by the nisA promoter.The theta replicon of the plasmid pRAF800 from L. lactis SMQ719 was amplified by PCR with the primers designed based on the pRAF800 sequence. The PCR products were digested with EcoRI and NheI and linked with the Em^r of pMG36e, then transformed into L. lactis NZ9000 by electroporation. The plasmid pEm^r:rep harboring the erythromycin resistance gene and theta replicon were screened on erythromycin plates. The repB-deficient companion plasmid pEm^r:â–³rep was constructed by enzyme digestion, deficiency and ligation, and could coextist with the plasmid with the same theta replicon in the media containing erythromycin only.