Study On The Role Of Na~+/K~+ Transporter In L-arginine Synthesis In Corynebacterium Crenatum

At present,with the increasing demand for L-arginine,there is increased interest in metabolic engineering of L-arginine-producing strains and application in microbial fermentation.However,the influence of ion transport has not been extensively studied.In the previous experiment,we found that appropriate addition of K⁺in the medium during fermentation was beneficial to the synthesis of L-arginine in Corynebacterium crenatum SYPA5-5.The main research results are as follows:?1?By transcriptome data analysis of C.crenatum SYPA5-5 in medium supplemented with 7mmol·L-¹ and 35 mmol·L-¹ K⁺,we found that the transcriptional level of cation transport ATPase CTAP1,CTAP2 and monovalent cation/H⁺antiporter Mrp1A,Mrp2C related genes were significantly increased by adding 35 mmol·L-¹ K⁺during L-arginine synthesis.It was found that Mrp1A is Na⁺/H⁺antiporter and Mrp2C is K⁺/H⁺antiporter,which could regulate ion homeostasis.CTAP1 and CTAP2 were specific K⁺transport ATPase,which could transport K⁺from the medium to intracellular environment,maintaining the intracellular ion homeostasis.The ion transporters are involved in ion homeostasis,providing physiological regulation basis for C.crenatum SYPA5-5 to adapt to the changes under different ion conditions.?2?To study the intracellular and extracellular ion changes,mutant strains were designed by knock out and overexpression of the genes mrp1,mrp2,ctap1 and ctap2 to construct mutant strains.When the strains were added with 35 mmol·L-¹ K⁺,the growth rates were significantly increased compared with that of 7 mmol·L-¹ K⁺.The strains could still grow after dilution of 10000 times.The absence of ion transporters Mrp1A led to the accumulation of intracellular[Na⁺]to 6649ppm·g^-11 DCW.Cells need to regulate intracellular ion homeostasis,leading to a decrease in intracellular[K+]concentration to 8000 ppm·g-1 DCW.From detecting intracellular pH of C.crenatum SYPA5-5 and mutant strains by BCECF-AM,we found that the intracellular pH of inactivated strains were alkaline but the overexpressed strains were neutral.?3?It was found that pyruvate kinase activity was highest when the concentration of K⁺was400-500 mmol·L-1,which was beneficial to ATP synthesis.At this time,the activities of pyruvate kinase of 5-5?ctap1ctap2?and 5-5?ctap1mrp1?reached 0.83 U·mL^-11 and 0.85 U·mL^-1,which increased 16.9%and 19.7%compared with C.crenatum SYPA5-5 respectively.The optimal concentration of PO₄^3-in C.crenatum SYPA5-5 was 25 mmol·L-¹.The activity of glucose6-phosphate dehydrogenase?G6PDH?in C.crenatum SYPA5-5 had the highest activity,which could reach 0.83 U·mL-¹.The activity of G6PDH was inhibited at higher PO₄^3-concentrations,which is not conducive to the production of L-arginine.However,the production of L-arginine was significantly increased after the addition of the same concentration of K3PO4.?4?When adding appropriate amount of potassium ions and phosphate ions to the medium,the L-arginine production of 5-5?mrp1?,5-5?ctap1?and 5-5?ctap1mrp1?in the fed-batch fermentation were 61.4 g·L-¹,63.9 g·L-¹ and 65.3 g·L-¹ respectively and the yields were 0.383 g·g^-1,0.392 g·g^-1and 0.395 g·g^-1.The strain overexpressing Mrp1A and CTAP1 was more beneficial to ion homeostasis,pH stability and osmotic pressure regulation,ultimately increasing the yield of L-arginine.The study of ion transporters Mrp1A,Mrp2C,CTAP1 and CTAP2 has laid the foundation for the relationship between ion transport mechanism and L-arginine synthesis.