Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remain...Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.展开更多
Annulus fibrosus(AF)repair remains a challenge because of its limited self-healing ability.Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair.Although the unique and be...Annulus fibrosus(AF)repair remains a challenge because of its limited self-healing ability.Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair.Although the unique and beneficial characteristics of decellularized extracellular matrix(ECM)in tissue repair have been demonstrated,the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration.In the present study,we combined polyethylene glycol diacrylate(PEGDA)and decellularized annulus fibrosus matrix(DAFM)to develop an injectable,photocurable hydrogel for AF repair.We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure.Transforming growth factor-β1(TGF-β1)was further incorporated into PEGDA/DAFM hydrogels,and it could be continuously released from the hydrogel.The in vitro experiments showed that TGF-β1 facilitated the migration of AF cells.Furthermore,PEGDA/DAFM/TGF-β1 hydrogels supported the adhesion,proliferation,and increased ECM production of AF cells.In vivo repair performance of the hydrogels was assessed using a rat AF defect model.The results showed that the implantation of PEGDA/DAFM/TGF-β1 hydrogels effectively sealed the AF defect,prevented nucleus pulposus atrophy,retained disc height,and partially restored the biomechanical properties of disc.In addition,the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue.In summary,findings from this study indicate that TGF-β1-supplemented DAFM hydrogels hold promise for AF repair.展开更多
Streptomycetes are Gram-positive bacteria with high GC DNA content. They produce the most abundant secondary metabolites including over two-thirds of the clinically used antibiotics of natural origin (Barka et al., 20...Streptomycetes are Gram-positive bacteria with high GC DNA content. They produce the most abundant secondary metabolites including over two-thirds of the clinically used antibiotics of natural origin (Barka et al., 2016), for example,the important broad-spectrum antimicrobials oxytetracycline(OTC) and chlortetracycline, which are the tetracycline antibiotics。展开更多
Coenzyme Q10(CoQ10)is an important component of the respiratory chain in humans and some bacteria.As a high-value-added nutraceutical antioxidant,CoQ10 has excellent capacity to prevent cardiovascular disease.The cont...Coenzyme Q10(CoQ10)is an important component of the respiratory chain in humans and some bacteria.As a high-value-added nutraceutical antioxidant,CoQ10 has excellent capacity to prevent cardiovascular disease.The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels.In this study,we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain.Moreover,under phosphate-limited conditions(decreased phosphate or in the absence of inorganic phosphate addition),CoQ10 production increased significantly by 12%to220 mg/L,biomass decreased by 12%,and the CoQ10 productivity of unit cells increased by 27%.In subsequent fed-batch fermentation,CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation.Furthermore,to understand the mechanism associated with CoQ10 overproduction under phosphate-limited conditions,the comparatve transcriptome analysis was performed.These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01.Phosphate limitation induced a pleiotropic effect on cell metabolism,and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.展开更多
Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces i...Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.展开更多
Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and ...Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm,but sensitive sensing and stable amplification in antigen detection remain challenging.Here,we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy.Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive,fast,and stable antigen detection.In a demonstration of this method,the limit of detection was at the single virus level(0.17 fM,approximately two copies/μL)in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples.The entire procedure required only 20 min.Given our system’s simplicity and modular setup,we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.展开更多
As synthetic biology enters the era of quantitative biology,mathematical information such as kinetic parameters of enzymes can offer us an accurate knowledge of metabolism and growth of cells,and further guidance on p...As synthetic biology enters the era of quantitative biology,mathematical information such as kinetic parameters of enzymes can offer us an accurate knowledge of metabolism and growth of cells,and further guidance on precision metabolic engineering.k_(cat),termed the turnover number,is a basic parameter of enzymes that describes the maximum number of substrates converted to products each active site per unit time.It reflects enzyme activity and is essential for quantitative understanding of biosystems.Usually,the k_(cat) values are measured in vitro,thus may not be able to reflect the enzyme activity in vivo.In this case,Davidi et al.defined a surrogate K^(vivo)_(max)(k_(app))for kcat and developed a high throughput method to acquire K^(vivo)_(max)from omics data.Heckmann et al.and Chen et al.proved that the surrogate parameter can be a good embodiment of the physiological state of enzymes and exhibit superior performance for enzyme-constrained metabolic model to the default one.These breakthroughs will fuel the development of system and synthetic biology.展开更多
The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and e...The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.展开更多
基金Science and Technology Research and Development Program of Shihezi University, No. ZRKX2009YB23
文摘Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.
基金the funding provided for this study by the National Natural Science Foundation of China(81925027,32130059,31872748,32171350,32101103)Natural Science Foundation of Jiangsu Province(BK20200199)+1 种基金China Postdoctoral Science Foundation(2021M702412)the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Annulus fibrosus(AF)repair remains a challenge because of its limited self-healing ability.Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair.Although the unique and beneficial characteristics of decellularized extracellular matrix(ECM)in tissue repair have been demonstrated,the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration.In the present study,we combined polyethylene glycol diacrylate(PEGDA)and decellularized annulus fibrosus matrix(DAFM)to develop an injectable,photocurable hydrogel for AF repair.We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure.Transforming growth factor-β1(TGF-β1)was further incorporated into PEGDA/DAFM hydrogels,and it could be continuously released from the hydrogel.The in vitro experiments showed that TGF-β1 facilitated the migration of AF cells.Furthermore,PEGDA/DAFM/TGF-β1 hydrogels supported the adhesion,proliferation,and increased ECM production of AF cells.In vivo repair performance of the hydrogels was assessed using a rat AF defect model.The results showed that the implantation of PEGDA/DAFM/TGF-β1 hydrogels effectively sealed the AF defect,prevented nucleus pulposus atrophy,retained disc height,and partially restored the biomechanical properties of disc.In addition,the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue.In summary,findings from this study indicate that TGF-β1-supplemented DAFM hydrogels hold promise for AF repair.
基金supported by the National Natural Science Foundation of China (31770055, 31922002, 31720103901, and 31772242)the 111 Project (B18022)+4 种基金the Fundamental Research Funds for the Central Universities (22221818014)the Shanghai Science and Technology Commission (18JC1411900)the Young Scientists Innovation Promotion Association of Chinese Academy of Sciences (2016087) to Weishan Wangthe Shandong Taishan Scholar Program of China to Lixin Zhangthe Open Project Funding of the State Key Laboratory of Bioreactor Engineering
基金supported by grants from Shengxue Dacheng Pharmaceutical Co.,Ltd in Shijiazhuang,Chinathe National Natural Science Foundation of China(31570031)
文摘Streptomycetes are Gram-positive bacteria with high GC DNA content. They produce the most abundant secondary metabolites including over two-thirds of the clinically used antibiotics of natural origin (Barka et al., 2016), for example,the important broad-spectrum antimicrobials oxytetracycline(OTC) and chlortetracycline, which are the tetracycline antibiotics。
基金The authors appreciate Dr.Jin Miao for the help to construct engineered strains in Table 1.The author also appreciates Prof.Hongwei Yu for providing plasmid materials.This work was supported by the National Natural Science Foundation of China[31870040,31430002,31720103901]the 111 Project of China[B18022]+2 种基金the Fundamental Research Funds for the Central Universities[22221818014]the Natural Science Foundation of Shandong Province[ZR2017ZB0206]the Shandong Taishan Scholar Award to Lixin Zhang.
文摘Coenzyme Q10(CoQ10)is an important component of the respiratory chain in humans and some bacteria.As a high-value-added nutraceutical antioxidant,CoQ10 has excellent capacity to prevent cardiovascular disease.The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels.In this study,we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain.Moreover,under phosphate-limited conditions(decreased phosphate or in the absence of inorganic phosphate addition),CoQ10 production increased significantly by 12%to220 mg/L,biomass decreased by 12%,and the CoQ10 productivity of unit cells increased by 27%.In subsequent fed-batch fermentation,CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation.Furthermore,to understand the mechanism associated with CoQ10 overproduction under phosphate-limited conditions,the comparatve transcriptome analysis was performed.These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01.Phosphate limitation induced a pleiotropic effect on cell metabolism,and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.
基金This work is supported by the Natural Science Foundation of Hebei Province(No.C2019209399)Tangshan Science and Technology Project(No.20130208b)+1 种基金the Science and Technology Program of Hebei(No.18222916)the Research Fund for Top Discipline Construction of North China University of Science and Technology(No.18060720),China.
文摘Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.
基金This work was supported by the National Key R&D program of China(2020YFA0907800)the National Natural Science Foundation of China(31922002,31720103901,31772242 and 31870040),the 111 Project(B18022)+1 种基金the Fundamental Research Funds for the Central Universities[22221818014]the Youth Innovation Promotion Association CAS(Y202027)to W.W and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm,but sensitive sensing and stable amplification in antigen detection remain challenging.Here,we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy.Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive,fast,and stable antigen detection.In a demonstration of this method,the limit of detection was at the single virus level(0.17 fM,approximately two copies/μL)in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples.The entire procedure required only 20 min.Given our system’s simplicity and modular setup,we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.
基金the National Natural Science Foundation of China(31922002,32101174)W.W and Z.L,the National Key R&D program of China(2020YFA0907800)+1 种基金the 111 Project(B18022),the Youth Innovation Promotion Association CAS(Y202027)W.W and the China Postdoctoral Science Foundation(2021M703379)to Z.L.
文摘As synthetic biology enters the era of quantitative biology,mathematical information such as kinetic parameters of enzymes can offer us an accurate knowledge of metabolism and growth of cells,and further guidance on precision metabolic engineering.k_(cat),termed the turnover number,is a basic parameter of enzymes that describes the maximum number of substrates converted to products each active site per unit time.It reflects enzyme activity and is essential for quantitative understanding of biosystems.Usually,the k_(cat) values are measured in vitro,thus may not be able to reflect the enzyme activity in vivo.In this case,Davidi et al.defined a surrogate K^(vivo)_(max)(k_(app))for kcat and developed a high throughput method to acquire K^(vivo)_(max)from omics data.Heckmann et al.and Chen et al.proved that the surrogate parameter can be a good embodiment of the physiological state of enzymes and exhibit superior performance for enzyme-constrained metabolic model to the default one.These breakthroughs will fuel the development of system and synthetic biology.
基金This work was supported by the National Natural Science Foundation of China[31870040]the National Key Research and Development Project(2020YFA0907804,2020YFA0907304)+1 种基金the“111”Project of China[B18022]the Fundamental Research Funds for the Central Universities[22221818014],and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.
