| L-arginine is an important amino acid which has a unique variety of physiological and pharmacological effects. Therefore, it has a very wide range of applications in medicine, food and other fields. Corynebacterium crenatum SYPA5-5 was a strain with a high yield of L-arginine production in our laboratory which was obtained by multi-level mutagenesis.One of the most important strategies to increase output of L-arginine is increasing the synthesis of the enzyme thus increasing L-arginine metabolic pathway flux. Whereas study on increasing the flux of amino acids of microbial cells found that this will lead to the accumulation of intracellular amino acids much higher than the original strain, indicating that the amino acid transport capability of recombination strain is very limited. Reducing the intracellular concentration of L-arginine to lift the feedback inhibition and feedback repression of its key enzyme in the synthesis metabolic pathway could help to improve the L-arginine metabolic pathway flux and thus increase its production. Through the transporting protein L-arginine excretes from inside to outside of cells. This paper studied the strain C. crenatum SYPA5-5 by gene disruption to verify the function of L-arginine transporter protein LysE in L-arginine fermentation, and we released L-arginine feedback inhibition from the perspective of the secretion of amino acid and strengthened expression of the L-arginine transporter protein to achieve the purpose of increasing L-arginine production.The L-arginine transporter protein LysE from Corynebacterium crenatum and ArgO from Escherichia coli are both transmembrane proteins. Studies of cloning of lysE gene in E. coli showed that the nucleotide sequences of lysE gene from C.crenatum shared a homology of 99.7% with the lysE gene from Corynebacterium glutamicum ATCC13032. The fusion expression vector pGEX-6P-1 could increase the expression of these two proteins. Western blot was applied to identify these two fusion-proteins.We constructed a plate-coloring method based on a L-arginine defective strain of E. coli for detecting fast and effectively L-arginine secretion qualitatively, and feasibility of its experimental results showed that the method used for the qualitative detection of the secretion of L-arginine was effective and feasible.In this paper we knocked the lysE gene out of C.crenatum SYPA5-5 successfully by use of suicide vector pK18mobsacB and obtained lysE deletion mutant△lysE/C.crenatum, and over-expression of LysE in this deletion mutant was acted to obtain pJC-tac-lysE/(△lysE/C.crenatum). The qualitative detection of△lysE/C.crenatum,pJC-tac-lysE/(△lysE/C.crenatum) and the original strain using the plate-coloring method and the fermentation experiments of these strains in basic medium verified initially the function of LysE in C.crenatum, that is, when the amount of L-arginine in C.crenatum cells exceeds a certain level LysE can transport intracellular L-arginine from inside to outside. Lacking of LysE could make L-arginine accumulation in the cells above the normal level, enhance the expression of LysE could, to some extent, weaken the the intracellular accumulation of L-arginine because of absence of LysE.Based on the study above, over-expression of lysE gene in C.crenatum SYPA5-5was done by introducing the recombinant shuttle plasmid pJC-tac-lysE. Further fermentation test of this recombination strain and the strains constructed above showed that compared with the original strain, the L-arginine production of pJC-tac-lysE/C.crenatum increased by 12% and the fermentation cycle has been shortened which indicates that over-expression of LysE in C. crenatum, to some extent, could increase L-arginine production. |
PDF Full Text Request