Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locu...Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway, ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA 1/A2 was positive regulatory element for red gene cluster.展开更多
Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying ...Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chro- mosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.展开更多
Thiols play very important role in the intracellular redox homeostasis. Imbalance in the redox status leads to changes in the intracellular metabolism including respiration. Thiol stress, a reductive type of stress ca...Thiols play very important role in the intracellular redox homeostasis. Imbalance in the redox status leads to changes in the intracellular metabolism including respiration. Thiol stress, a reductive type of stress can also cause redox imbalance. When Gram-positive bacterium Strep- tomyces coelicolor was exposed to thiol stress, catalaseA was induced. Induction of catalaseA is the consequence of elevation of ROS (reactive oxygen species). The two major sources of reactive oxygen species are Fenton reaction and slippage of electrons from electron transport chain during respiration. Hence, the effect of thiol stress was checked on the rate of oxidative phosphorylation in S. coelicolor. We found correlation in the increase of oxidative phosphorylation rate and the generation of ROS, subsequently leading to induction of catalase. It was observed that thiol stress does not affect the functionality of the individual complexes of the ETC, but still there was an increase in the overall respiration, which may lead to generation of more ROS leading to induction of catalase.展开更多
Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramaticall...Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g^(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.展开更多
Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites.Osmoregulation is important for their lifecycle in nature.In the genome of ...Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites.Osmoregulation is important for their lifecycle in nature.In the genome of Streptomyces coelicolor M145,SCO3128(encodes a putative fatty acid desaturase),SCO3129(encodes a putative TetR family regulator)and SCO3130(encodes a putative L-carnitine dehydratase)constitute a transcriptional unit,and its transcript was found to be in response to osmotic stress.Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium.These results indicated that SCO3130 is important for the osmotic stress resistance in S.coelicolor.Transcriptional analysis and electrophoretic mobility shift assays(EMSA)demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128,indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.展开更多
A new gene, scrX from Streptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons in scrX which are AAA, AAA and ATA. scrXgene may...A new gene, scrX from Streptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons in scrX which are AAA, AAA and ATA. scrXgene may be a typical differentiation regulator which was strictly controlled at translational level. The comparison of amino acids also revealed that ScrX belonged to Id R family which acted in transcriptional regulation of prokaryote. Studies on gene function by gene disruption and complementation indicated that scrX may play a positive regulation role in spore formation of Streptomyces coelicolor.A new gene, scrX from Streptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons in scrX which are AAA, AAA and ATA. scrXgene may be a typical differentiation regulator which was strictly controlled at translational level. The comparison of amino acids also revealed that ScrX belonged to Id R family which acted in transcriptional regulation of prokaryote. Studies on gene function by gene disruption and complementation indicated that scrX may play a positive regulation role in spore formation of Streptomyces coelicolor.展开更多
Over past few years various protocols have been developed to produce gold nanorods by chemical synthesis but, most of the chemical used in synthesis are toxic which shows immense efect on environment. In the present i...Over past few years various protocols have been developed to produce gold nanorods by chemical synthesis but, most of the chemical used in synthesis are toxic which shows immense efect on environment. In the present investigation, we developed a simple bio-based synthesis of gold nanorods employing blue pigment produced by Streptomyces coelicolor klmp33 using microwave irradiation. The pigment used to reduce HAuCl4 in solution to yield gold nanorods with in 120 s. The gold nanorods were characterized by UV-visible spectroscopy; transmission electron microscopy（TEM）, X-ray difraction（XRD） and Fourier transform infrared spectroscopy（FTIR）. The bio-based synthesis developed in this method is safe, eco-friendly, rapid and appropriate way for bulky synthesis of gold nanorods.展开更多
The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this ...The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.展开更多
Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources.Undecylprodigiosin(UP)is a promising antibiotic,but the direct f...Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources.Undecylprodigiosin(UP)is a promising antibiotic,but the direct feeding of pure precursor amino acids makes it costly for large-scale production.Here,collagen peptide(CP),a renewable animal-derived biomass contains abundant precursor amino acids of UP.CP can act as carbon and nitrogen source for the growth of Streptomyces coelicolor CGMCC 4.7172.The plant biomasses including soybean meal,wheat bran,and malt extract were unsuitable for UP prodution.However,365.40µg/L UP was detected after 24 h in the media containing CP,and its highest concentration reached 1198.01µg/L.UP was also detected in the media containing meat hydrolysates of domestic animals,but its initial production time was delayed,and final concentration was lower than that in the medium containing CP only.Compared the fermentation performances of CP and other proteins,CP has a special superiority for UP production.These results revealed that UP biosynthesis may be dependent on amino acid availability of substrates and CP is beneficial for UP production because of its specific amino acid composition.展开更多
文摘Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway, ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA 1/A2 was positive regulatory element for red gene cluster.
基金Project (No. 2004-527) supported by the Scientific Research Foun-dation for the Returned Overseas Chinese Scholars, State EducationMinistry, China
文摘Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chro- mosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.
文摘Thiols play very important role in the intracellular redox homeostasis. Imbalance in the redox status leads to changes in the intracellular metabolism including respiration. Thiol stress, a reductive type of stress can also cause redox imbalance. When Gram-positive bacterium Strep- tomyces coelicolor was exposed to thiol stress, catalaseA was induced. Induction of catalaseA is the consequence of elevation of ROS (reactive oxygen species). The two major sources of reactive oxygen species are Fenton reaction and slippage of electrons from electron transport chain during respiration. Hence, the effect of thiol stress was checked on the rate of oxidative phosphorylation in S. coelicolor. We found correlation in the increase of oxidative phosphorylation rate and the generation of ROS, subsequently leading to induction of catalase. It was observed that thiol stress does not affect the functionality of the individual complexes of the ETC, but still there was an increase in the overall respiration, which may lead to generation of more ROS leading to induction of catalase.
基金supported by the National Natural Science Foundation of China(31430004,31421061,31630003,31370081 and 31570072)the Science and Technology Commission of Shanghai Municipality(16490712100)
文摘Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g^(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.
基金We are grateful to Prof.Keith Chater(John Innes Centre,Norwich,UK)for providing E.coli ET12567(pUZ8002)Prof.Keqian Yang(Institute of Microbiology,CAS)for pIMEP.This work was supported by grants from the National Natural Science Foundation of China(No.31170088).
文摘Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites.Osmoregulation is important for their lifecycle in nature.In the genome of Streptomyces coelicolor M145,SCO3128(encodes a putative fatty acid desaturase),SCO3129(encodes a putative TetR family regulator)and SCO3130(encodes a putative L-carnitine dehydratase)constitute a transcriptional unit,and its transcript was found to be in response to osmotic stress.Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium.These results indicated that SCO3130 is important for the osmotic stress resistance in S.coelicolor.Transcriptional analysis and electrophoretic mobility shift assays(EMSA)demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128,indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.
文摘A new gene, scrX from Streptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons in scrX which are AAA, AAA and ATA. scrXgene may be a typical differentiation regulator which was strictly controlled at translational level. The comparison of amino acids also revealed that ScrX belonged to Id R family which acted in transcriptional regulation of prokaryote. Studies on gene function by gene disruption and complementation indicated that scrX may play a positive regulation role in spore formation of Streptomyces coelicolor.A new gene, scrX from Streptomyces coelicolor was cloned and sequenced. It consists of 660 base pair, encoding a peptide of 220 amino acids. There are three rare codons in scrX which are AAA, AAA and ATA. scrXgene may be a typical differentiation regulator which was strictly controlled at translational level. The comparison of amino acids also revealed that ScrX belonged to Id R family which acted in transcriptional regulation of prokaryote. Studies on gene function by gene disruption and complementation indicated that scrX may play a positive regulation role in spore formation of Streptomyces coelicolor.
文摘Over past few years various protocols have been developed to produce gold nanorods by chemical synthesis but, most of the chemical used in synthesis are toxic which shows immense efect on environment. In the present investigation, we developed a simple bio-based synthesis of gold nanorods employing blue pigment produced by Streptomyces coelicolor klmp33 using microwave irradiation. The pigment used to reduce HAuCl4 in solution to yield gold nanorods with in 120 s. The gold nanorods were characterized by UV-visible spectroscopy; transmission electron microscopy（TEM）, X-ray difraction（XRD） and Fourier transform infrared spectroscopy（FTIR）. The bio-based synthesis developed in this method is safe, eco-friendly, rapid and appropriate way for bulky synthesis of gold nanorods.
基金supported by the National Natural Science Foundation of China(Grant Nos.30400259,30221003)the National Basic Research Program(973 Program)(Grant No.2007CB914301)the Tianjin Municipal Science and Technology Commission(Grant No.08SYSYTC00200).
文摘The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
基金We acknowledge the financial support provided by the National Key R&D Program of China(2017YFB0308500).
文摘Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources.Undecylprodigiosin(UP)is a promising antibiotic,but the direct feeding of pure precursor amino acids makes it costly for large-scale production.Here,collagen peptide(CP),a renewable animal-derived biomass contains abundant precursor amino acids of UP.CP can act as carbon and nitrogen source for the growth of Streptomyces coelicolor CGMCC 4.7172.The plant biomasses including soybean meal,wheat bran,and malt extract were unsuitable for UP prodution.However,365.40µg/L UP was detected after 24 h in the media containing CP,and its highest concentration reached 1198.01µg/L.UP was also detected in the media containing meat hydrolysates of domestic animals,but its initial production time was delayed,and final concentration was lower than that in the medium containing CP only.Compared the fermentation performances of CP and other proteins,CP has a special superiority for UP production.These results revealed that UP biosynthesis may be dependent on amino acid availability of substrates and CP is beneficial for UP production because of its specific amino acid composition.
