Breeding Of L-glutamic Acid Producing Strains With Extensive PH Adaptability By Genome Shuffling

Glutamic acid is a typical traditional fermentation product. In its fermentation control, pH is one important parameter. Thus, it is of great value for industrial application to screen for some glutamic acid producing strains with extensive pH adaptability, for they help to reduce the consumption of ammonia and concentrated sulfuric acid, and alleviate the adverse effects of extensive operations on production in fermentation. In this research, genome shuffling was used to breed glutamic acid producing strain with high tolerance for acid and alkali. Four strains with good production capacity were obtained after two rounds of genome shuffling, and furthermore DNA polymorphism comparison between the mutants and the original strain was performed by AFLP (amplified fragment length polymorphism) analysis.First, nine glutamic acid producing strains, which were preserved in our lab, have been compared in tolerance to low pH, high pH and fermentation characteristics. SW07-01, SW07-02 and T613-85 have higher tolerance for low pH while SW07-01, SW07-03 and T613-85 perform well in the high pH conditions and SW07-01, SW07-06 have the highest yield of glutamate acid. In conclusion, SW07-01 was selected for further study.Second, a rapid high-throughput screening method based on fermentation in tissue culture plate has been established as follows: the mutated strains were pre-screened in the extreme pH plates, then single colonies were transfered to the fermentation medium (containing pH indicator) plates, afterwards the strains with color circle were inoculated into tissue culture plates for fermentation. Finally the ones producing more glutamic acid than 95% of the original strain's were selected for the finanl screening via shake-flask fermentation.SW07-01 was cultured for 5 h, and then mutated by DES or X ray treatment. After screening, four strains with low pH tolerance and three with high pH tolerance were obtained. The seven mutants were performed genome shuffling as parents. First round generated three mutants with extensive pH adaptability, designated as F1-1-1,F1-1-4 and F1-2-2, while second round performed with the F1 strains resulted in four mutants F2-1-17, F2-2-7, F2-8-18 and F2-9-11.The original strain, mutants, F1 and F2 strains have been compared in tolerance to acid, alkali and fermentation characteristics. In every aspect, same tendency, F2 > F1 > mutants > SW07-01, was observed. SW07-01 could grow at pH value ranging from 5.0 to 10.8, while F2 could grow from pH 4.6 to pH 11.4. In the shake-flask fermenation, F2 yielded 76.7% more glutamic acid than SW07-01 and 23.0% more in the 5 L fermentor.DNA polymorphism of F2 strains and SW07-01 were compared by using AFLP method. The cluster result showed that genetic similarity coefficient of F2-2-7 and SW07-01 is 0.93.