Rational Transformation Of Metabolic Engineering And Optimization Of Fermentation Control Of L-Glutamic Acid Producer

L-glutamate is one of the basic amino acids for nitrogen metabolism in organisms,and plays an important role in the metabolism of substances in life.Due to its unique physiological function,glutamic acid is widely used in food,medicine,chemical industry,cosmetics and feed industries,and has become the world’s largest amino acid with broad market prospects.At present,glutamic acid fermentation with biotin suboptimal strains have problems such as low acid production,low sugar-to-acid conversion rate,insufficient bacterial activity,and easy aging and autolysis during the fermentation process.In view of the above problems,this study is based on the metabolic engineering,using Corynebacterium glutamicum GDK-9 as the starting strain to carry out genetic engineering transformation,and at the same time to develop and optimize its fermentation process,so as to improve Glutamate yield and sugar acid conversion rate.The main research contents are as follows:Corynebacterium glutamicum GDK-9 was used as the starting strain,and the exogenous phosphoketolase gene—xfp was introduced into Corynebacterium glutamicum GDK-9.At the same time,the expression intensity of it and ppc gene was optimized,which alleviated the problem that pyruvate generates acetyl Co A and releases CO₂,resulting in carbon loss.Finally,genetically engineered bacteria GX-4 was obtained.This strain and the starting strain were verified by 5 L fermentation.The results showed that the L-glutamic acid yield of the GX-4 strain was 136 g/L,an increase of 18.2%,and the lactic acid and L-alanine contents were 4.8 g/L and 4.8 g/L,respectively.3.7 g/L,decreased by 7.7%and 9.8%,sugar-acid conversion rate was 69.4%,increased by 3.8%.In order to improve the bacterial activity and L-glutamic acid production during glutamic acid fermentation,the effects of rare earth elements on glutamic acid fermentation were investigated.Three rare earth elements La³⁺,Ce³⁺and Nd³⁺were selected,and the optimum addition amount of glutamic acid fermentation was obtained by single factor experiment and orthogonal experiment.On this basis,the addition method was optimized,and finally it was concluded that the optimal use method of rare earth elements was continuous flow addition for 10 h after the start of fermentation,and the flow addition amount was 100 mg/L La³⁺,9 mg/L Ce³⁺and 0.6 mg/L Nd³⁺.The fermentation results showed that the maximum bacterial cell mass was 56,the L-glutamic acid production was 148 g/L,which were increased by 11.1%and 11.3%,respectively,and the sugar-acid conversion rate was72.3%,which was increased by 2.9%.The whole nutrient fed-batch strategy was adopted to solve the problems of insufficient bacterial activity and decreased acid production capacity in the late stage of L-glutamic acid fermentation.Through experimental analysis,it was determined that the optimal fed-feeding conditions were fed-feeding medium with a volume fraction of 60%from 2 h of fermentation,and fed-feeding time was 24 h.After verification by the 5 L fermenter,the OD_600nm reached66,an increase of 29.4%,and the cell transformation time was advanced from 4 h to 2 h.The L-glutamic acid yield was 168 g/L,an increase of 22.6%.The sugar-acid conversion rate was72.3%,an increase of 2.9%.In order to alleviate the problem that the high concentration of NH₄⁺inhibits the activity of bacteria due to the adjustment of p H by stream addition of ammonia water during L-glutamic acid fermentation,a nitrogen source two-stage fermentation control process was proposed.It was found in the experiment that the mixing ratio of Na OH and ammonia water was 1:7 at the beginning of fermentation,and the mixing ratio of Na OH and ammonia water was 1:3 to replace ammonia water for p H adjustment after 19 h of fermentation,which had the best improvement effect.Under these conditions,the final yield of L-glutamate was 171g/L,increased by 11.8%,and the conversion rate of sugar and acid also increased from 72.3%to 74.1%.The metabolic flow analysis of L-glutamate showed that the metabolic flow of L-glutamate increased by 3.9%.Based on rational transformation of metabolic engineering,this study combined with rare earth element addition,total nutrient flow addition process and nitrogen source two-stage fermentation control process to achieve a breakthrough in l-glutamate yield and sugar and acid conversion rate,which has reference significance for the refinement control and efficient acid production of glutamate fermentation industry.