After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of...After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.展开更多
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
文摘After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.
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.
