Production of L-citrulline by a recombinant Corynebacterium crenatum SYPA 5-5 whole-cell biocatalyst / 生物工程学报

Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.

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）

<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>

Expression and characterization of ArgR, an arginine regulatory protein in Corynebacterium crenatum / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.</p><p><b>METHODS</b>Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.</p><p><b>RESULTS</b>Arginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.</p><p><b>CONCLUSION</b>The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.</p>