Breeding Of Strain Highly Producing Phenyllactic Acid By Genome Shuffling

Phenyllactic acid, PLA is a natural antimicrobial compounds, which has the efficacy to preserve fresh food from rotting;also as a derivative of Danshensu Salvianic acid A, PLA has the same pharmacological effects in vitro anti-platelet aggregation activity, in the food and pharmaceutical fields, its market prospect is very broad. In this study, genome shuffling was used to breed the producing strain to improve the PLA production rate of fermentation to promote the development of the industry, increase the social and economic benefits. The traditional mutation breeding techniques and microbial protoplast fusion were successfully reorganized by Gene technology for combination to the reorganization of objects from a single gene to the entire genome, while many parent strains were excellent phenotype with a reorganization of the strains,which could be efficiently and quickly filter out the fermentation of mutant strains for positive good character, instead of the mutants that is difficult to improve the performance of the defects after a long tradition of mutagen glazing.Firstly, a strain Lactobacillus paracasei W2 producing phenyllactic acid was isolated from the yogur brought, which grew well in MRS medium, inhibited the ability of food spoilage. The production of lactic acid could be increased by adding the ppa in the culture medium, the fermentation results show that the optimal concentration of ppa was 4 g/L, while the highest yield of PLA is 0.81g/L.The strain was identified by the physiological and biochemical method and 16SrDNA,sequenced as Lactobacillus paracasei, the GenBank accession number is HM101340.Lactobacillus paracasei W2,as parental strain, was mutagenized UV-ray and NTG.10 mutants were selected by the isolation on the calcium carbonate plates and Oxford Cup inhibition experiments from141 survival mutants. As a result of fermentation in Erlenmeyer flask, U4, U7, U12, and N3, N6,were more increased of Pla producing compared with the original mutant.These mutants were cultured for forward 10 generations, each generation of strains detected in the fermentation broth of Pla, little change saved with genetic stability. The optimum age for the mutation strain was 18-20h,by determining the growth curve of Lactobacillus paracasei W2.The genome shuffing was used to improve the production of phenyllactic acid by Lactobacillus paracasei W2. The starting population (U4,U7,U12,N3,N6) mutated by ultraviolet irradiation and nitrosoguanidine were used as the parental strains of protoplast fusion. After several rounds of genome shuffing, a forward mutant F8 was screened, its phenyllactic acid production was 1.92mg/ml,which increased 1.37 times more than the original strain.The optimum concentration of mutanolysin is 15μg/ml and the lysozyme is 20mg/mL. The optimum conditions of protoplast fusion were found, room temperature,PEG50%,the time of protoplast fusion is 10min.