• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.312-319
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• 2002

The Sporosarcina psychrophilia pyrB gene, which encodes aspartate transcarbamylase (ATcase), was cloned on Sau3AI restriction endonuclease fragment inserted into pUC19 plasmid vector, S. psychrophilia pyrB gene was expressed in Escherichia coli pyrB mutant for the complementation test. The sequence of 2,606 nucleotides including putative pyrB gene was determined. The region contained one full open reading frame (ORf) and two partial ORFs. The deduced amino acid sequence of the second ORF showed 59% identity with that of Bacillus caldolyticus ATCase. The first and third partial ORFs were closely related to the uracil permease (pyrP) and dihydroorotase (pyrC), respectively. Besides, potential terminator, antiterminator, and anti-antiterminator structures were found in the intergenic region between pyrP and pyrB. These results suggested that S. psychrophilia pyrimidine nucleotide biosynthesis genes are clustered as well as other Bacillus sp. Over-expressed product of pyrB encoding ATCase was purified and analyzed by the SDS-PAGE. The purified PyrB protein turned out to be molecular mass of 27 kDa and showed ATCase activity.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• KSBB Journal
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• v.9 no.4
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• pp.372-377
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• 1994

To improve L-lysine yield, pyrimidine base analogue(6-azauracil)-resistant mutants were isolated from Corynebacterium glutamicum KFCC10672 Among them the best producer, C. glutamicum CH0516, was selected and tested for L-lysine production in a $7\ell$ fermentor. It was found that the product yield obtained with C. glutamicum CH0516 was higher than that of the parent strain by 3%. In order to elucidate the gain in productivity with the 6-azauracil-resistant mutant enzymatic kinetic parameters such as aspartokinase(AKase) and aspartate carbamoyltransferase (ATCase) were measured. The Km values of AKase with C. glutamicum KFCC10672 and CH0516 were 200.0 mM and 166.7 mM and those of ATCase were 0.13 mM and 0.27 mM, respectively. However, the specific enzyme activities of AKase of C. glutamlcum KFCC10672 and CH0516 were $3.89{\times}10^{-1}$ units/mg and $4.78{\times}10^{-1}$ units/mg, and those of ATCarse were 2.20 units/mg and 1.84 units/mg, respectively. It appears that some increase in product yield with C. gluramicum CH0516 is likely due to the increased Akase activity and decreased ATCase activity.