Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and proB Gene Deletion in Corynebacterium crenatum / 生物医学与环境科学(英文)
Biomedical and Environmental Sciences
;
(12): 864-874, 2015.
Artigo
em Inglês
| WPRIM
| ID: wpr-258866
ABSTRACT
<p><b>OBJECTIVE</b>In Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.</p><p><b>METHODS</b>NAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.</p><p><b>RESULTS</b>The IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.</p><p><b>CONCLUSION</b>Feedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Arginina
/
Prolina
/
Mutagênese Sítio-Dirigida
/
Deleção de Genes
/
Fosfotransferases (Aceptor do Grupo Carboxila)
/
Corynebacterium
/
Retroalimentação Fisiológica
/
Escherichia coli
/
Genética
/
Metabolismo
Limite:
Animais
Idioma:
Inglês
Revista:
Biomedical and Environmental Sciences
Ano de publicação:
2015
Tipo de documento:
Artigo
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