Isolation And Identification Of Glutathione- Containing Lactic Acid Bacteria And Construction Of Glutathione-Producing Recombinant Lactic Acid Bacteria

Glutathione(GSH) has many important physiological functions like immunity improvement, free radicals scavenging, detoxification. It also plays an important role in repairing and synthesizing of protein, synthesizing of DNA and the transportation of amino acids. Considering that, it is of urgent to screen strains with high GSH content, to find novel bifunctional GSH synthesis enzyme, and to construct strains with high GSH yield, especially probiotic Lactic acid bacteria.In order to get high GSH containing probiotic Lactic acid bacteria, a strain with high GSH content was screened. It was studied with physiological and biochemical analysis, molecular biology analysis, and bioinformatics analysis systematically. The potential gshF gene was cloned and expressed in prokaryote heterologously. Based on that, a recombinant Lactic acid bacteria was constructed with the ability to absorb and synthesize GSH. Main conclusions are summarized as follows:With commercially available yogurt fermentation agent, Inner Mongolia cheese and pickles, a strain with high content of GSH was screened, and its GSH content reached 6.14 mg/g(dry cell weight). The strain was identified as Lactobacillus paracasei by cultural characteristics, physiological and biochemical property as well as phylogenetic status analysis, and it was named Lactobacillus paracasei LHA4.The potential gshF gene was cloned and constructed into expression system of E. coli and L. lactis. Sequence analysis showed that similarity between GshFLp and GshF from Pasteurella multocida 2000 and Streptococcus thermophilus CNRZ1066 was only 27%and 25%. And GshFLp was shorter than GshF with catalytic activity by 100 amino acids. Two recombinant engineering strain, E. coli BL21/pET28a-gshFLp and L. lactis NZ9000/pNZ8148-gshFLp, were constructed to investigate the function of GshFLp. After induction with IPTG and nisin, GshFLp was expressed in E. coli BL21 and L. lactis NZ9000. However, neither pure GshFLp from E. coli BL21 nor crude enzyme of L. lactis NZ9000/pNZ8148-gshFLp.was found of function. At the same time, crude enzyme of L. paracasei LHA4 also failed to synthesize GSH with Glu, Gly and Cys. All in all, we concluded that the potential GshFLp didn’t have the ability to synthesize GSH.To further enhance the content of GSH in L. paracasei, we selected genome of S. thermophilus as the template and cloned the bifunctional GSH synthesis gene, gshFst-After that, it was constructed into expression ventor pMG36e and introduced to L. paracasei LHA4 with electroporation to get L paracasei LHA4/pMG36e-gshFst. Results show that GSH content in the engineering strain increased by 56% than the wild strain.