Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth p...Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.展开更多
A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization...A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.展开更多
基金Supported by the National Natural Science Foundation of China（20536040） the State Key Development Program for Basic Research of China（2007CB707802） the Development Project of Science and Technology of Tianjin（05YFGZGX04500）
文摘Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.
文摘A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.
