Construction Of Lactoccus Lactis Expression Systenm Based On ThyA Gene And Cloning Of Nattokinase Gene

The lactic acid bacteria (LAB) is a clade of gram-positive bacteria which canproduce lactic acid as the major metabolic end-product of carbohydrate fermentation，andthey are generally regarded as safe food microorganism．The species Lactococcus lactis, whichbecame the best studied LAB in this group, in particular was demonstrated to be an idealcell factory for heterogenous proteins after Escherichia coli and Bacillus subtilis. In orderto select the appropriate transformants and maintain selective pressure on the geneticmodification, these gene cloning vectors and expression systenms were equipped with oneor more genes coding for resistance to antibiotics. As the container of resistance genes, thestrains can transfer these antibiotic resistance encoding genes to other symbiotic bacteriaand that will threaten the biological safety. So using food-grade selection markers toreplace the antibiotic selectable ones has become research hotspot in all the world.Thymidine synthase gene thyA widely exists in the phage, prokaryotic and eukaryotic cellsand it is the key enzyme to denovo biosynthesis of dTMP. Therefor, thyA-null strainscouldn’t multiply because of DNA biosynthesis is restricted, unless add thymine orthymidine to the medium or transform complementary plasmid containing whole sequenceof thyA gene into them.In this study, In order to enhance the transposition frequency of IS1, we used extremeglucose levels (4%) and streptomycin below minimum inhibitory concentration(1.25μg/mL) to culture of E. coli DH5α, and then obtained the thyA-null mutant E. coli D8bythe improved method of trimethoprim (TMP) directional selection. A whole IS1was insertin the open reading frame of thyA gene, and insertion site was the118bp after the initiationcodon. The sequence from109bp to118bp (AACCTGCAAG) generated duplication of aten base pair sequence and that was the fisrt report of course10bp duplication at theinsertion site. The method lay the foundation for constrcting nutritional deficient strain byusing endogenous insertion sequence. The thyA-null mutant E. coli D8could restore theability to grow on the minimal medium without thymine by using exogenous thyA gene.D8could be a host of food-grade vector which use thyA gene as selected marker and thatfacilitate the gene manipulation. As a result, D8complement the Lactoccus.lactisexpression systenm based on thyA gene. thyA gene was cloned from Streptococcus thermophilus St-JY, which was isolatedfrom yogurt, and the thyA gene was used to replace the erythromycin resistance genes onLactoccus.lactis expression vector pMG36e to construct a food-grade vector pMGthyA.The new vector only contains a replicon from cryptic plasmid pWVO1ofLactoccus.lactis, a thyA gene from Streptococcus thermophilus St-JY and a multiplecloning site from pUC18.Amylase gene amy of Bacillus licheniformis was cloned on the MCS of the vectorpMGthyA as a reporter gene, yielded a recombinant plasmid pMGthyA-amy. The plasmidwas electroporated into thyA-null host L.lactis MG1363-TT and the transformants couldbe selected on minimal medium, SA medium. The recombination strain on SA platescontaining0.5%(w/v) soluble starch could form obvious bacterial colony and the clearzones (halo) around the colonies could be observed after added iodine solution.Furthermore, the nattokinase gene fragment pre-pro-NK (ppNK) ecoding signal peptide,propetide and mature peptide, the fragment pro-NK (pNK) ecoding propetide and maturepeptide, and the fragment NK ecoding mature peptide were amplified by PCR fromBacillus subtilis var. natto genomic DNA and cloned on the MCS of pMGthyA respectively.Then the three recombinant plasmids were electroporated into L.lactis MG1363-TT. Ourresearch findings lay the foundation for food-grade expression nattokinase in L.lactis.