Molecular Breeding Of Corynebacterium Crenatum For L-arginine Fermentation Production

L-arginine, an important semi-essential amino acid, participates in the urea cycle as a precursor for nitric monoxide, and plays an important role in maintaining physiological functions in human and animals. Currently, L-arginine is primarily produced by microbial fermentation, and thus breeding of arginine high-yield strains has become a focus in industry.C. crenatum MT argB M4 with ArgR lethal mutation is a mutant strain in which a key rate-limiting enzyme: N-acetyl-L-glutamate kinase(NAGK) of L-arginine biosynthesis was constructed to be less sensitive to L-arginine feedback inhibition by site-directed mutagenesis. In this work, C. crenatum MT argB M4 ?proC, C. crenatum MT argB M4 ?proB, C. crenatum MT argB M4 ?pta, C. crenatum MT argB M4 ?ack, C. crenatum MT argB M4 ?cgl2310, C. crenatum MT argB M4 ?proB ?cgl2310, and C. crenatum MT argB M4 ?proB ?cgl2310 ?pta were separately constructed based on genome-scale metabolic network model(GSMN) of Corynebacterium glutamicum for improvement of arginine production of the mutant strain. Aseries of shake flask fermentation was carried out and main results were showed as follows:1. The final L-arginine production of C. crenatum MT argB M4 ?proB was significantly increased by approximately 19.6 % higher than that of the original strain, but the growth rate and glucose consumption were decreased. When 10 mM L-proline were added to the fermentation medium, the final L-arginine production of C. crenatum MT argB M4 ?proB was 16.45 g/L, significantly increased by approximately 34.8 % higher than that of the original strain, meanwile its growth rate and glucose consumption was the same as the original strain. Additionally, with 10 mM L-proline added to the medium, the final L-arginine production of C. crenatum MT argB M4 ?pro C was increased by 13.1 % higher than that of the original strain. Therefore, knockout proB is more reasonable than knockout proC.2. The growth was stimulated by acetate synthesis pathway elimination, and the glucose consumption had no change. In addition, the final L-arginine production of C. crenatum MT argB M4 ?pta or C. crenatum MT argB M4 ?ack was significantly increased by approximately 27 % or 13.1 % higher than that of the original strain. Therefore, knockout pta is more reasonable than knockout ack.3. Without any change of the growth rate and glucose consumption, the final L-arginine production of C. crenatum MT argB M4 ?cgl2310 was significantly increased by approximately 27.5 % higher than that of the original strain.4. With neglectable change of the growth rate and glucose consumption, the final L-arginine production of C. crenatum MT argB M4 rproB rcgl2310 rpta was reached 21.7 g/L and was significantly increased by approximately 16 % higher than that of C. crenatum MT argB M4 rproB rcgl2310 and 31.5 % higher than that of C. crenatum MT argB M4 ?proB.