The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four...The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose-synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acs operon sequence of A. xylinum ATCC 53582. Alignment of the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302-3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen-tube-pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.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
Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase woul...Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.展开更多
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
Participation of Pseudomonas putida-derived methyl phenol（dmp） operon and Dmp R protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentatio...Participation of Pseudomonas putida-derived methyl phenol（dmp） operon and Dmp R protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has Dmp R protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon,phenol hydroxylase encoded by dmp N gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from Dmp R protein and of the DNA sequences from the two Upstream Activation Sequences（UAS）present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation.展开更多
Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants...Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.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
Impact statement Arsenic is the most common toxic metalloid in the environment.Nearly all organisms have genes for arsenic detoxification.Arsenic detoxification genes are frequently organized in chromosomal or plasmid...Impact statement Arsenic is the most common toxic metalloid in the environment.Nearly all organisms have genes for arsenic detoxification.Arsenic detoxification genes are frequently organized in chromosomal or plasmid-encoded arsenic resistance(ars)operons,which are commonly regulated by members of the ArsR transcriptional repressors.To date,three As(Ⅲ)-responsive ArsRs with different As(III)binding sites have been identified.Here,we identify a new type of As(Ⅲ)-responsive ArsR repressor that has an atypical As(Ⅲ)binding site and controls transcription of the ars operon of Arsenicibacter rosenii SM-1.Our results provide new insights into the classification and evolution relationship of the ArsR transcriptional repressors.展开更多
Escherichia coli contains 12 chaperone-usher operons,including 64 genes,used for biosynthesis and assembly of various fimbriae which consume a lot of energy and material.In this study,each of the 12 operons was delete...Escherichia coli contains 12 chaperone-usher operons,including 64 genes,used for biosynthesis and assembly of various fimbriae which consume a lot of energy and material.In this study,each of the 12 operons was deleted in an L-threonine-producing E.coli strain TWF001,and the resulting 12 deletion mutants produced more L-threonine than TWF001 after 16 or 24 h cultivation.Therefore,the 12 chaperone-usher operons were deleted in different combinations,resulting in 11 strain mutants which lack at least 2 operons.The cell growth and L-threonine production of these 11 mutants were determined.Among these 11 mutants,TWK021 in which 10 chaperone-usher operons were deleted,showed the highest L-threonine production.TWK021 produced 15.75 g L-threonine from 40 g glucose after 36 h cultivation.The conversion rate of glucose to L-threonine reached 0.394 g/g in TWK021,which is 32.2%higher than the control strain TWF001.These results suggest that the fimbria lacking E.coli TWK021 is a good host for efficient production of L-threonine.展开更多
OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount ...OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.展开更多
Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding...Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position –81 to –68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position –49 to –36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (?5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are re- quired for PurR binding to the pur operon control site.展开更多
Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the r...Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule.In this study,we first report a possible example of the DnaA‐dependent riboswitch for transcription attenuation in Escherichia coli.We show that(i)DnaA regulates the transcription of the structural genes but not that of the leader hisL gene;(ii)DnaA might bind to rDnaA boxes present in the HisL‐SL RNA,and subsequently attenuate the transcription of the operon;(iii)the HisL‐SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important;and(iv)the translating ribosome is required for deattenuation of the his operon,whereas tRNA^(His) strengthens attenuation.This mechanism seems to be phylogenetically conserved in Gram‐negative bacteria and evolutionarily important.展开更多
文摘The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose-synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acs operon sequence of A. xylinum ATCC 53582. Alignment of the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302-3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen-tube-pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.
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.
基金supported by the National Key Project (2009CB941601, 2008ZX08006-004 and 2009ZX0890-024B)the Joint Funds of the National Natural Science Foundation of China (u0731004)
文摘Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.
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deeply indebted to DST-PURSE program 2012–2015 going on in Department of Biochemistry and Biophysics, University of Kalyani for providing different equipments and essential infrastructural supportDeep gratitude is extended to DBT sponsored Bioinformatics Infrastructure Facility in the Department of Biochemistry and Biophysics, University of Kalyani for the necessary support
文摘Participation of Pseudomonas putida-derived methyl phenol（dmp） operon and Dmp R protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has Dmp R protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon,phenol hydroxylase encoded by dmp N gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from Dmp R protein and of the DNA sequences from the two Upstream Activation Sequences（UAS）present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation.
基金supported by the National Natural Scientific Foundation(No.81201251)from the Ministry of Science and Technology of the People’s Republic of Chinathe Priority Project on Infectious Disease Control and Prevention(No.2012ZX10004215 and 2013ZX10004-610-007)from the Ministry of Health and the Ministry of Science and Technology of the People’s Republic of Chinathe Science Foundation for the State Key Laboratory for Infectious Disease Prevention and Control from China(Grant No.2015SKLID508 and 2011SKLID202)
文摘Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.
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.
基金supported by the Natural Science Foundation of China(Grant Nos.42077210 and 41930758 to Ke Huang and Fang-jie Zhao,respectively).
文摘Impact statement Arsenic is the most common toxic metalloid in the environment.Nearly all organisms have genes for arsenic detoxification.Arsenic detoxification genes are frequently organized in chromosomal or plasmid-encoded arsenic resistance(ars)operons,which are commonly regulated by members of the ArsR transcriptional repressors.To date,three As(Ⅲ)-responsive ArsRs with different As(III)binding sites have been identified.Here,we identify a new type of As(Ⅲ)-responsive ArsR repressor that has an atypical As(Ⅲ)binding site and controls transcription of the ars operon of Arsenicibacter rosenii SM-1.Our results provide new insights into the classification and evolution relationship of the ArsR transcriptional repressors.
基金supported by the National Key Research and Development Program of China(2021YFC2100900).
文摘Escherichia coli contains 12 chaperone-usher operons,including 64 genes,used for biosynthesis and assembly of various fimbriae which consume a lot of energy and material.In this study,each of the 12 operons was deleted in an L-threonine-producing E.coli strain TWF001,and the resulting 12 deletion mutants produced more L-threonine than TWF001 after 16 or 24 h cultivation.Therefore,the 12 chaperone-usher operons were deleted in different combinations,resulting in 11 strain mutants which lack at least 2 operons.The cell growth and L-threonine production of these 11 mutants were determined.Among these 11 mutants,TWK021 in which 10 chaperone-usher operons were deleted,showed the highest L-threonine production.TWK021 produced 15.75 g L-threonine from 40 g glucose after 36 h cultivation.The conversion rate of glucose to L-threonine reached 0.394 g/g in TWK021,which is 32.2%higher than the control strain TWF001.These results suggest that the fimbria lacking E.coli TWK021 is a good host for efficient production of L-threonine.
基金ThisstudywassupportedbyagrantfromtheNationalHighTechnologyResearchandDevelopmentProgram (No .10 2 0 8 0 20 2)
文摘OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.
基金This work was supported by the National Natural Science Foundation of China(Grant No.30070419).
文摘Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position –81 to –68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position –49 to –36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (?5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are re- quired for PurR binding to the pur operon control site.
基金supported by grants from the National Natural Science Foundation of China(Grant no.32260233 to Morigen)the Science and Technology Foundation of Inner Mongolia(Inner Mongolia Key Laboratory for Molecular Regulation of the Cell,Grant no.2021PT0002).
文摘Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule.In this study,we first report a possible example of the DnaA‐dependent riboswitch for transcription attenuation in Escherichia coli.We show that(i)DnaA regulates the transcription of the structural genes but not that of the leader hisL gene;(ii)DnaA might bind to rDnaA boxes present in the HisL‐SL RNA,and subsequently attenuate the transcription of the operon;(iii)the HisL‐SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important;and(iv)the translating ribosome is required for deattenuation of the his operon,whereas tRNA^(His) strengthens attenuation.This mechanism seems to be phylogenetically conserved in Gram‐negative bacteria and evolutionarily important.
