Breeding Of High-yield Glutamic Acid-producing Strains By Heavy Ion Beam Irradiation And The Mechanism Study Of Amoxicillin-induced Fermentation

Glutamate is the most abundant amino acid in the world and has a wide range of applications in food,medicine,industry and agriculture.Fermentation is an important method for industrial production of glutamic acid.At present,the production cost of glutamic acid is high and the level that production of glutamic acid is low.It can effectively reduce the cost of production and increase the profits of enterprises through increasing the yield of glutamic acid and the glutamate conversion yield.It's still unclear that the mechanism of antibiotic-induced glutamate secretion at the transcriptional level,even though a large number of studies on the mechanism of glutamate overproduction at home and abroad.According to the metabolic regulation strategy,we try to irradiate Corynebacterium glutamicum by heavy ion beam,then irradiated strains were screened by culture through fermentation in flasks and acquire high-glutamic acid produce mutant stain.At the same time,the mechanism of glutamate secretion induced by amoxicillin was preliminarily explored.Screening of mutants after irradiation mutagenesis using high-sugar,malonic acid,sulfaguanidine and other resistant plates,the result is selecting 73 strains with high glucose resistance mutant strain,malonic acid 101 strains of resistant mutant strain and 89strains of sulfa-resistant mutant strain;the selected mutant strains was subjected to select by24-well plate fermentation and saved,and then we try to rescreen by shake flask to obtain a high-acid-producing mutant strain with an acid production rate of 90.25.g/L,which increased about 9.1%more than before.The single-factor experiment was used to optimize the fermentation medium,and it was found that glucose,corn steep and K₂HPO₄ had a greater impact on glutamate production.Response surface experiment optimization component was glucose 188g/L,corn steep liquor 6.2g/L,K₂HPO₄1.7g/L.After execute 4 experiments,the glutamate yield was close to the average of 98.5 g/L,which was close to the predicted value.The mechanism of the effect of amoxicillin on glutamate secretion was studied by measuring the growth status of the strain,the content of glutamic acid and the relative expression levels of key genes in the fermentation process.It is found that the increasing of cell membrane?wall?permeability is a necessary condition for the secretion of glutamate;in the late stage of glutamic acid production,excessive biotin can enhance the transcription level of key genes involved in glutamate synthesis and secretion,including:pc?pepc?cs?icdh?icl?gdh?pdh?ldh?msccG?msccG2?odhA.Amoxicillin can reduce the transcription level of whcD and ACCase,and then the cell membrane?wall?can not be synthesized normally,and the permeability of the cell membrane?wall?is enhanced.Above all,this condition can activate the transport protein and allow glutamate to be secreted to the outside.