Breeding Of L-Isoleucine-Producing Strain And Studies On Its Fermentation Conditions

According to the theory of metabolic control fermentation, the dissertation focuses on the breeding and fermentation conditions of L-isoleucine producing strain. The main research contents and results are as follows:(1) The method of paper chromatograghy-mottle elution chromometry was defined to determine L-isoleucine concentration quantitatively in the fermentation broth. The application conditions of this method were studied.(2) One L-isoleucine high-producing strain WYJl(Met"+AECr+ a -AB1) was derived from Brevibacterium flavum TL411 by stepwise mutagenic treatments with DBS and UV, which could produce L-isoleucine 8.2 g/L under the fermentation condition without being optimized.(3) The batch fermentation conditions of strain WYJ1 in shake flask were studied in this dissertation. The proportions of seed medium and fermentation medium components were optimized by uniform design experiment and MATLAB software .The seed culture conditions and fermentation conditions were also studied.Under the optimum conditions, strain WYJ1 could produce L-isoleucine 16.7g/L after fermentation for 96 hours by flask-shaking batch fermentation.(4) The metabolic flux analysis (MFA) was applied in L-isoleucine fermentation process based on the experimental data from batch fermentation in 5-liter fermentor. Based on the flux balance principle, the metabolic flux analysis method of L-isoleucine batch fermentation was defined .The whole metabolic process of L-isoleucine was analysed combined with fermentation process curve. Using metabolic flux balance model of L-isoleucine biosynthesis by Brevibacterium flavum , the metabolic flux distributions during the middle and late period of fermentation process were determined and the optimal flux distributions and all kinds of extreme flux distributions were calculated by linear program of MATLAB software .The flux analysis indicated that a large number of NADPH were useful to He biosynthesis; The proper activity ratio of PPC/PDC ,the genetic manipulation and fermentation control for reducing the concentration of amino acid byproduct were important for L-isoleucine high productivity, as well as the optimum soluble oxygen control.The mutant WYJ1 obtained has been potential to be applied to industrial producing, which could produce L-isoleucine 18.3g/L in 5L fermentor and whose genetic character was stable. The application of metabolic flux analysis on L-isoleucine fermentation in 5L fermentor would promote the furthermoredevelopment of this mutant and optimize the metabolic flux of L-isoleucine biosynthesis.