Beijing Corynebacterium Glutamicum Yielding Mutation Breeding

Corynebacterium pekinense has been used extensively for the industrial productionof L-glutamate (Glu), which is an important fermentation product. In this thesis, byprotoplasts mutation with UV radition, a mutant strain with higher glutamate productionability was obtained. The optimal condition for protoplasts preparation was that the cellcultivating8h was dealt with1.5g·/l glycin and0.2U·/l penicillin for2h and thendigested with2×10~4U·/l lysozyme at35℃for8h. By plate count method, the formationand the regeneration rate of protoplasts reached97.93%and10.48%, respectively. Theprotoplasts were radiated by UV at15W and with a distance of25cm for45min. Thelethality of the protoplasts attained97.93%. Under this condition, a mutant strain S4with higher glutamate production ability and higher inherit stability was selected in aplate with high substrate and product concentration. In the flask culture of semisyntheticmedium, the glutamate reached41.3g·/l. Compared with the original strain, the productconcentration, yield, and biomass of the mutant strain S4was increased by35%,4%and8.5%, respectively.The optimization of culture medium for S4in batch fermentation was carried outby single factorial and response surface experiments. The results showed suitablecomposition was: glucose80g/l, urea2.2g/l, phosphate3.6g/l and yeast extract3g/l.The analysis of variance indicated the interaction between urea and phosphate is notsignificant. The interaction between yeast extract and phosphate are very significant andthe interaction between yeast extract and urea are significant. By monitoring theconcentrations of urea and phosphate in the fermentation process, it was found3.6g/lphosphate, which is only used in the first24h and kept the same level in the wholefermentation process, is enough for the fed-batch fermentation. And the consumption ofurea is cell growth dependent. To obtain higher cell concentration, more urea and yeastextract should be added with glucose with a ratio of0.15g yeast extract+0.1g urea per 10g glucose in fed-batch fermentation.With the increase of initial glucose in fermentation system, substrate inhibitionappeared to be significant and productivity of glutamate decreased gradually. Becauseof the high cost of glutamate recovery from fermentation broth, economic production ofglutamate from glucose requires the improvement of both product concentration andproductivity. Glutamate fermentation was inhibited by high glucose concentration, sofed-batch cultivations with low substrate concentrations were studied to maximize thefinal product concentration and productivity. The results showed that the optimal culturecondition was the mixed substrate of nutrients and glucose. The experimental resultsshowed that this feeding mode could provide a better cell growth environment andimprove the cell concentration. This feeding strategy could give68.4g/l glutamate witha productivity of0.66g/l·h and a yield of0.62g/g, which was increased by21.3％and22.2%compared with that of glucose pulse feeding strategies.