| Corynebacterium glutamicum is an aerobic,non-pathogenic gram-positive bacterium.It has been widely used in the industrial production of L-lysine.During the fermentation of L-lysine,many by-product amino acids are produced,such as glutamic acid,methionine,leucine,isoleucine and valine.Thus,reducing the accumulation of by-products not only facilitates the downstream purification and recovery process of L-lysine,as the carbon source is more concentrated for the synthesis of the target product and may further increase the yield of the target product.Studies have found that the extracellular transport of amino acids requires the help of corresponding amino acid transporters,and the effect of the lack of related transporters on the synthesis of L-lysine has not yet been reported.In this paper,C.glutamicum 23604 was used as the starting strain.Using molecular biology methods,based on the principle of two homologous single exchanges,three kinds of amino acid transporters were inactivated by antibiotic resistant plate screening and sucrose plate rescreening.The effect of gene deletion on the synthesis of C.glutamicum 23604 L-lysine was analyzed.The main results were as follows:1.The knockout vector pK19 mobsacB-gluE was used to knock out the gluE gene of glutamate extracellular transporter.The recombinant bacterium C.glutamicum WL1701 was cultured in minimal medium for 96 hours and its glutamic acid was detected.The yield was reduced by 69.7% compared with the control bacteria,indicating that the ability of C.glutamicum 23604 to secrete glutamic acid was greatly reduced but not completely lost after knocking down the gluE gene.It is speculated that there may be other extracellular glutamate transport pathways in C.glutamicum 23604.The study also found that the L-lysine production of C.glutamicum WL1701 in minimal medium was improved to some extent compared to the control bacteria and increased by 9% in the fermentation medium,indicating that the deletion of gluE causes a decrease in the amount of glutamic acid,while allowing the intracellular carbon source to accumulate and flow in the direction of producing L-lysine.2.The knockout vector pK19 mobsacB-brnFE was used to knock out the brnFE gene of branched amino acids and methionine extracellular transporter.The recombinant bacterium C.glutamicum WL1702 was cultured in minimal medium for 96 hours.Compared with the control strains,the production of valine,leucine and isoleucine were all reduced to 0 mg/L,and the production of methionine was reduced by 63.7%.After knocking out the brnFE gene,the amount of byproduct amino acids produced by the C.glutamicum WL1702 decreased significantly.At the same time,it was found that the L-lysine production of C.glutamicum WL1702 in minimal medium was somewhat improved compared to the control bacteria.In the fermentation medium,the biomass of the recombinant strain C.glutamicum WL1702 was decreased by 12.7% in the stationary phase compared with the control strain,and the glucose consumption rate was significantly faster than that of the control.The final yield of L-lysine increased by 12.3% compared to the control bacteria.This shows that the loss of the brnFE gene results in a reduction in the biomass of the cells,an increase in the rate of glucose consumption,and a decrease in the production of by-product amino acids,resulting in a greatly increased ability to produce L-lysine in a single strain.3.The lysine intracellular transporter gene lysP was knocked out by the gene knockout vector pK19mobsacB-lysP.The yield of L-lysine was higher in the culture medium C.glutamicum WL1703 cultured for 96 hours.The control strain increased by 10%.After analysis of the fermentation broth,it was found that,except for the large difference in the content of L-lysine,the types and contents of other amino acids were approximately the same.Therefore,after lysP was knocked out,it did not affect other amino acids and only affected L-lysine production,indicating that LysP is a more specific amino acid transporter.4.To analyze the synergistic relationship between multiple channel proteins and the effect on the yield of L-lysine,a double deletion strain,C.glutamicum WL1704 and C.glutamicum WL1706,and a three deletion strain,C.glutamicum WL1705,were constructed.After cultured in the fermentation medium,the lysine yields of the two double-deleted bacteria were only increased by 3% and 2% compared with the control bacteria,but they were somewhat lower than those of the single-deleted bacteria.The L-lysine production of theC.glutamicum WL1705 was reduced by 10.8% compared to the control bacteria.The reason may be that the excessive deletion of the channel protein on the cell membrane leads to the imbalance of intracellular and extracellular ion transport,thereby decreasing the yield of L-lysine.It is also possible that a variety of by-product amino acids are accumulated intracellularly together to produce synergistic feedback inhibition,inhibiting an enzyme on the common pathway for amino acid synthesis,and thereby reducing the yield of L-lysine. |
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