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

According to the theory of metabolic control fermentation and metabolic flux analysis, the dissertation focuses on the breeding of L-valine high-yielding strain and its fermentation conditions. The main research contents and results are as follows:(1) L-valine concentration in fermented broth was quantitatively determined with methods of paper chromatography-mottle elution colorimetry and chemical colorimetry. The quantitative determination condition and computing method were decided. Compared with high-speed amino acid analyzer, the determination value of L-valine concentration with chemical colorimetry is more accurate than with paper chromatography-mottle elution colorimetry.(2) The optimum condition of protoplast ultraviolet mutation was determined. Brevibacterium flavum TV 10 as a starting strain was stepwise mutated with protoplast UV and Diethyl sulfate. One L-valine high-producing strain TV2564 with genetic marker was screened through shake tube primary screening, shake flask compound screening, genetic marker test and single clone isolation and continuous passage cultivation. This strain could produce L-valine 29.3 Og/L after fermentation for 72 hours under the fermentation condition without being optimized. Genetic character of strain TV2564 was very stable.(3) The batch fermentation condition of strain TV2564 in shake flask was studied in this dissertation. The seed medium was optimized by neural network and genetic algorithms, and the seed culture conditions were also studied. Based on the single factor experiment of fermentation medium, three main factors were screened by the Placket-Burman design method. Then, the best level of three factors were determined by response surface analysis and the fermentation condition was studied. Under the optimum condition, the strain TV2564 could produce L-valine 39.5g/L after fermentation for 72 hours by flask-shaking batch fermentation.(4) The metabolic network was proposed for L-valine producing strain. The metabolic flux distributions of Brevibacterium flavum TV2564 during the different fermentation period were calculated by linear program of MATLAB software according to metabolic flux analysis theory. The metabolic flux distributions of BMP, HMP, TCA cycle and the key principle nodes of GLC6P, PEP and PYR were analyzed. At the same time it can be indicated to provide theory guide for the fermentation process.(5) Based on the above optimum condition, the batch fermentation was performed with strain TV2564 in 5-liter fermentor. According to the change of DOin 5L fermentor, with dynamics analysis and characteristics of metabolic flux distributions during fermentation process, two stages oxygen supply was provided and it has been experimentally verified that the mutant could produce L-valine 45.1 g/L after fermentation for 64 hours. The L-valine batch fermentation kinetics was studied by neural network based on the experimental data from 5-liter fermentor's batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.(6) Fed batch fermentation was studied with TV2564 according to the optimum condition of batch fermentation. The optimum initial glucose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24th, 36th, 48th, 60th hour respectively. The production of L-valine reached 42.2g/L after fermentation for 70 hours. Based on the above experiment, fed batch fermentation in 5-liter fermentor was performed. Because of the technology of low glucose, low amino and fed batch, production of L-valine reached 50.71 g/L after fermentation for 60 hours. The production was improved by 20.16% and 12.43% than that of fermentation in shake flask fed batch and 5-liter fermentor batch.The innovation of this dissertation in the research field of L-valine metabolic control fermentation is that protoplast UV technique was firstly applied to breed L-valine high-yielding strain. An L-v...