Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor ...Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor regeneration,the high-volume input and limited cofactor recyclability still make the enzymatic processes unsustainable.Therefore,it is of great significance to reduce cofactor input in a fusion enzyme(FuE)system,but no successful practice has been reported.Herein,we design a decapeptide bridge,RRRQRRRARR(R10),with high affinity for NADPH to construct fusion oxidoreductases(phenylacetone monooxygenase and phosphite dehydrogenase)for ester synthesis and NADP recycling.The peptide bridge enables electrostatic cofactor channeling that transports NADPH/NADP^(+)across the peptide between the enzymes’NADP-binding pockets,so the fusion enzyme(FuE-R10)presents 2.1-folds and 2.0-folds higher conversions than mixed free enzymes and a flexible linker(GGGGSGGGGS)-fused enzyme,respectively,at NADPH/FuE of 0.1.The fusion enzyme,FuE-R5,bridged by a half-shortened linker,is proved more effective in facilitating cofactor channeling;compared to the mixed free enzymes,FuE-R5 exhibits two orders of magnitude reduction of NADPH input in ester synthesis.The work has thus demonstrated the potential of the cofactor bridging strategy in the development of sustainable cofactor-dependent cascade biocatalysis.展开更多
Pre-harvest sprouting(PHS)or vivipary is a major problem affecting cereal quality and grain quantity and is primarily linked to the dysregulation of abscisic acid(ABA)biosynthesis in plants.Therefore,elucidating the m...Pre-harvest sprouting(PHS)or vivipary is a major problem affecting cereal quality and grain quantity and is primarily linked to the dysregulation of abscisic acid(ABA)biosynthesis in plants.Therefore,elucidating the molecular mechanisms governing seed dormancy is crucial for developing strategies to improve crop productivity.In this study,we identified a novel viviparous maize mutant,viviparous-like 5(vp-like5),which exhibits precocious germination in developing seeds.Through map-based cloning,we discovered that ZmCNX6,which encodes a small subunit of molybdopterin synthase essential for molybdenum cofactor(MoCo)biosynthesis,is the causal gene responsible for the vp-like5 phenotype.Biochemical assays have demonstrated significantly reduced activities of MoCo-dependent enzymes,including aldehyde oxidase(AO),xanthine dehydrogenase(XDH),and nitrate reductase(NR),in vplike5.AO is essential for the ABA biosynthesis,and the observed ABA deficiency in vp-like5 likely drives the viviparous phenotype.Expression analysis showed that ZmCNX6 was stably expressed during seed development,indicating its significant role in seed development.Furthermore,overexpression of ZmCNX6 not only enhanced the activities of MoCo-dependent enzymes but also improved drought tolerance in maize.Collectively,our study revealed ZmCNX6 as a multifunctional hub coordinating MoCo metabolism,ABA-dependent dormancy regulation,and abiotic stress responses,offering a potential target for simultaneously mitigating vivipary and improving drought resistance in maize.展开更多
Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-p...Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.展开更多
The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence...The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.展开更多
Two k-hyperconnection expressions of a general k-order cofactor Y<sub>（i,j）</sub> are presentedfor the indefinite parameter matrix Y of a linear system by applying directed hypergraph theory,and based on...Two k-hyperconnection expressions of a general k-order cofactor Y<sub>（i,j）</sub> are presentedfor the indefinite parameter matrix Y of a linear system by applying directed hypergraph theory,and based on it a decomposition theorem of Y<sub>（i,j）</sub> is derived.By this theorem,the multi-leveltearing and analysis can be carried out easily for any linear large system.This is a new mul-tilevel topological analysis method.Using proposed method the scale of systems which can betopologically analysed by a computer will be enlarged.展开更多
Introduction: High grade dysplasia of the cervix has a high incidence and can progress to cervical cancer. The aim was to study cofactors associated with high-grade cervical dysplasia. Methodology: This was a retrospe...Introduction: High grade dysplasia of the cervix has a high incidence and can progress to cervical cancer. The aim was to study cofactors associated with high-grade cervical dysplasia. Methodology: This was a retrospective case-control study without matching. Women with high grade dysplasia were the cases while those with a normal screening test represented the controls. The study took place at the Gabriel Touré University Hospital Center in Bamako. We included 351 cases and 420 controls. The capture and analysis were performed using the SPSS 20 software. A univariate and multivariate logistic regression analysis was performed for the analysis of risk cofactors. The statistical tests used were the odds ratio and its confidence interval and the statistical significance threshold was set at p Results: In univariate analysis, the co-factors statistically significantly associated with the occurrence of high-grade dysplasia were parity 0.6 (0.5 - 0.9), gestational 0.7 (0.5 - 0.9), smoking of the spouse 3.4 (1.1 - 11.3), the non-schooling 1.4 (1.2 - 2.1). In multivariate analysis after adjusting for confounding factors, two co-factors have significantly increased the risk of high-grade dysplasia: lack of schooling 1.4 (1.2 - 2.0) and polygamy 1.5 (1.4 - 2.5). Conclusion: At the end of this study, polygamy and lack of schooling were the main risk factors. The prevention of cervical cancer will go through the education of girls and women as well as communication for behavioral change and social change.展开更多
Microtubules play important roles in neuronal morphogenesis, including cellular polarization, neurite growth, and branching. A microtubule is a polymer of a- and β-tubulin heterodimers that are formed by a multistep ...Microtubules play important roles in neuronal morphogenesis, including cellular polarization, neurite growth, and branching. A microtubule is a polymer of a- and β-tubulin heterodimers that are formed by a multistep process assisted by at least five tubulin-fold- ing cofactors （TBCA-E） （Lopez-Fanarraga et al., 2001）. Newly syn- thesized α- and β-tubulins associate with the cytosolic chaperonin complex （CCT）, and then the quasi-native tubulins interact with five tubulin-folding cofactors. The α- and β-tubulins first interact with TBCB and TBCA, which are further transferred to TBCE and TBCD, respectively. The α- and β-tubulins, TBCC, TBCD, and TBCE form a supercomplex, and guanosine triphosphate （GTP） hydrolysis occurs within the supercomplex, releasing a tubulin heterodimer. In addition to their functions in tubulin folding and tubulin heterodimer formation, excess amounts of TBCD or TBCE may interact with tubulin heterodimers, leading to their degrada- tion. Thus, tubulin-folding cofactors may play a role in both the synthesis and degradation of tubulin heterodimers.展开更多
In literature, neutral polyethylene oxide (PEO) flocculated fines at low shear rates, while with cofactor (CF) addition, the formed CF-PEO complex showed larger ability to bridge fines, producing flocs. In this work, ...In literature, neutral polyethylene oxide (PEO) flocculated fines at low shear rates, while with cofactor (CF) addition, the formed CF-PEO complex showed larger ability to bridge fines, producing flocs. In this work, some process factors were found having significant effects on fines flocculation. Increases in CF to PEO ratio at constant PEO enhanced the bridging bonds, causing increases in flocculation initial rate (efficiency), amplitude (floc size), and fastness (a decrease in characteristic time). On the other hand, an increase in stirring rate (shear rate) in flocculation vessel caused decreases in initial rate and amplitude, and an increase in the fastness. All runs showed transient flocculation;the amplitude increased with time, reached maximum at equilibrium, and then started to decrease showing deflocculation. In brief, the CF to PEO ratio and the shear rate were found important parameters in mill operation, having significant effects on flocculation efficiency, fastness, and floc size.展开更多
Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide ad...Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent biosynthesis.However,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent biosynthesis.Herein,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334.NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH activity.Using this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed libraries.We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.展开更多
A T-mesh is basically a rectangular grid that allows T-junctions. Recently, Deng etal introduced splines over T-meshes, which are generalizations of T-splines invented by Sederberg etal, and proposed a dimension formu...A T-mesh is basically a rectangular grid that allows T-junctions. Recently, Deng etal introduced splines over T-meshes, which are generalizations of T-splines invented by Sederberg etal, and proposed a dimension formula based on the B-net method. In this paper, we derive an equivalent dimension formula in a different form with the smoothing cofactor method.展开更多
Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds.Here,the combination of optimizing precursor synthesis and balanc...Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds.Here,the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli.First,glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001,resulting in the best candidate strain TS-1.Then icd and gdhA were overexpressed in TS-1,which led to the accumulation of 1.060 g/L tryptophan.Subsequently,one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-D-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine,resulting in the accumulation of 1.380 g/L tryptophan.Finally,to maintain cofactor balance,sthA and pntAB,encoding transhydrogenase,were overexpressed.With sufficient amounts of precursors and balanced cofactors,the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation,which was 2.76-times higher than the titer of the parent strain.Taken together,our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan.Similar strategies could be applied to the production of other amino acids or related derivatives.展开更多
Hematopoiesis requires finely tuned regulation of gene expression at each stage of development.The regulation of gene transcription involves not only individual transcription factors(TFs)but also transcription complex...Hematopoiesis requires finely tuned regulation of gene expression at each stage of development.The regulation of gene transcription involves not only individual transcription factors(TFs)but also transcription complexes(TCs)composed of transcription factor(s)and multisubunit cofactors.In their normal compositions,TCs orchestrate lineage-specific patterns of gene expression and ensure the production of the correct proportions of individual cell lineages during hematopoiesis.The integration of posttranslational and conformational modifcations in the chromatin landscape,nucleosomes,histones and interacting components via the cofactor-TF interplay is critical to optimal TF activity.Mutations or translocations of cofactor genes are expected to alter cofactor-TF interactions,which may be causative for the pathogenesis of various hematologic disorders.Blocking TF oncogenic activity in hematologic disorders through targeting cofactors in aberrant complexes has been an exciting therapeutic strategy.In this review,we summarize the current knowledge regarding the models and functions of cofactor-TF interplay in physiological hematopoiesis and highlight their implications in the etiology of hematological malignancies.This review presents a deep insight into the physiological and pathological implications of transcription machinery in the blood system.展开更多
文摘Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor regeneration,the high-volume input and limited cofactor recyclability still make the enzymatic processes unsustainable.Therefore,it is of great significance to reduce cofactor input in a fusion enzyme(FuE)system,but no successful practice has been reported.Herein,we design a decapeptide bridge,RRRQRRRARR(R10),with high affinity for NADPH to construct fusion oxidoreductases(phenylacetone monooxygenase and phosphite dehydrogenase)for ester synthesis and NADP recycling.The peptide bridge enables electrostatic cofactor channeling that transports NADPH/NADP^(+)across the peptide between the enzymes’NADP-binding pockets,so the fusion enzyme(FuE-R10)presents 2.1-folds and 2.0-folds higher conversions than mixed free enzymes and a flexible linker(GGGGSGGGGS)-fused enzyme,respectively,at NADPH/FuE of 0.1.The fusion enzyme,FuE-R5,bridged by a half-shortened linker,is proved more effective in facilitating cofactor channeling;compared to the mixed free enzymes,FuE-R5 exhibits two orders of magnitude reduction of NADPH input in ester synthesis.The work has thus demonstrated the potential of the cofactor bridging strategy in the development of sustainable cofactor-dependent cascade biocatalysis.
基金supported by the Open Project of Key Laboratory,Xinjiang Uygur Autonomous Region(2023D04070)the Agricultural Science and Technology Innovation Program of CAAS(CAAS-CSCB-202403)。
文摘Pre-harvest sprouting(PHS)or vivipary is a major problem affecting cereal quality and grain quantity and is primarily linked to the dysregulation of abscisic acid(ABA)biosynthesis in plants.Therefore,elucidating the molecular mechanisms governing seed dormancy is crucial for developing strategies to improve crop productivity.In this study,we identified a novel viviparous maize mutant,viviparous-like 5(vp-like5),which exhibits precocious germination in developing seeds.Through map-based cloning,we discovered that ZmCNX6,which encodes a small subunit of molybdopterin synthase essential for molybdenum cofactor(MoCo)biosynthesis,is the causal gene responsible for the vp-like5 phenotype.Biochemical assays have demonstrated significantly reduced activities of MoCo-dependent enzymes,including aldehyde oxidase(AO),xanthine dehydrogenase(XDH),and nitrate reductase(NR),in vplike5.AO is essential for the ABA biosynthesis,and the observed ABA deficiency in vp-like5 likely drives the viviparous phenotype.Expression analysis showed that ZmCNX6 was stably expressed during seed development,indicating its significant role in seed development.Furthermore,overexpression of ZmCNX6 not only enhanced the activities of MoCo-dependent enzymes but also improved drought tolerance in maize.Collectively,our study revealed ZmCNX6 as a multifunctional hub coordinating MoCo metabolism,ABA-dependent dormancy regulation,and abiotic stress responses,offering a potential target for simultaneously mitigating vivipary and improving drought resistance in maize.
基金supported by the National Key R&D Program of China(2019YFA0904900)the National Natural Science Foundation of China(21877112,21837002,21721004)。
文摘Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.
文摘The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.
文摘Two k-hyperconnection expressions of a general k-order cofactor Y<sub>（i,j）</sub> are presentedfor the indefinite parameter matrix Y of a linear system by applying directed hypergraph theory,and based on it a decomposition theorem of Y<sub>（i,j）</sub> is derived.By this theorem,the multi-leveltearing and analysis can be carried out easily for any linear large system.This is a new mul-tilevel topological analysis method.Using proposed method the scale of systems which can betopologically analysed by a computer will be enlarged.
文摘Introduction: High grade dysplasia of the cervix has a high incidence and can progress to cervical cancer. The aim was to study cofactors associated with high-grade cervical dysplasia. Methodology: This was a retrospective case-control study without matching. Women with high grade dysplasia were the cases while those with a normal screening test represented the controls. The study took place at the Gabriel Touré University Hospital Center in Bamako. We included 351 cases and 420 controls. The capture and analysis were performed using the SPSS 20 software. A univariate and multivariate logistic regression analysis was performed for the analysis of risk cofactors. The statistical tests used were the odds ratio and its confidence interval and the statistical significance threshold was set at p Results: In univariate analysis, the co-factors statistically significantly associated with the occurrence of high-grade dysplasia were parity 0.6 (0.5 - 0.9), gestational 0.7 (0.5 - 0.9), smoking of the spouse 3.4 (1.1 - 11.3), the non-schooling 1.4 (1.2 - 2.1). In multivariate analysis after adjusting for confounding factors, two co-factors have significantly increased the risk of high-grade dysplasia: lack of schooling 1.4 (1.2 - 2.0) and polygamy 1.5 (1.4 - 2.5). Conclusion: At the end of this study, polygamy and lack of schooling were the main risk factors. The prevention of cervical cancer will go through the education of girls and women as well as communication for behavioral change and social change.
基金supported by grants from the Ministry of Education,Culture,Sports,Science and Technology in Japan to MM and TCthe Japan Society for the Promotion of Science to MO,MM,and TCthe Japan Science and Technology Agency to M.M.and TC
文摘Microtubules play important roles in neuronal morphogenesis, including cellular polarization, neurite growth, and branching. A microtubule is a polymer of a- and β-tubulin heterodimers that are formed by a multistep process assisted by at least five tubulin-fold- ing cofactors （TBCA-E） （Lopez-Fanarraga et al., 2001）. Newly syn- thesized α- and β-tubulins associate with the cytosolic chaperonin complex （CCT）, and then the quasi-native tubulins interact with five tubulin-folding cofactors. The α- and β-tubulins first interact with TBCB and TBCA, which are further transferred to TBCE and TBCD, respectively. The α- and β-tubulins, TBCC, TBCD, and TBCE form a supercomplex, and guanosine triphosphate （GTP） hydrolysis occurs within the supercomplex, releasing a tubulin heterodimer. In addition to their functions in tubulin folding and tubulin heterodimer formation, excess amounts of TBCD or TBCE may interact with tubulin heterodimers, leading to their degrada- tion. Thus, tubulin-folding cofactors may play a role in both the synthesis and degradation of tubulin heterodimers.
文摘In literature, neutral polyethylene oxide (PEO) flocculated fines at low shear rates, while with cofactor (CF) addition, the formed CF-PEO complex showed larger ability to bridge fines, producing flocs. In this work, some process factors were found having significant effects on fines flocculation. Increases in CF to PEO ratio at constant PEO enhanced the bridging bonds, causing increases in flocculation initial rate (efficiency), amplitude (floc size), and fastness (a decrease in characteristic time). On the other hand, an increase in stirring rate (shear rate) in flocculation vessel caused decreases in initial rate and amplitude, and an increase in the fastness. All runs showed transient flocculation;the amplitude increased with time, reached maximum at equilibrium, and then started to decrease showing deflocculation. In brief, the CF to PEO ratio and the shear rate were found important parameters in mill operation, having significant effects on flocculation efficiency, fastness, and floc size.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110201)the National Key R&D Program of China(2018YFA0901500)+3 种基金the National Natural Science Foundation of China(32070083 and 32222004)the Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00017)the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008).
文摘Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent biosynthesis.However,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent biosynthesis.Herein,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334.NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH activity.Using this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed libraries.We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.
文摘A T-mesh is basically a rectangular grid that allows T-junctions. Recently, Deng etal introduced splines over T-meshes, which are generalizations of T-splines invented by Sederberg etal, and proposed a dimension formula based on the B-net method. In this paper, we derive an equivalent dimension formula in a different form with the smoothing cofactor method.
基金This work was supported by the National Key R&D Program of China(2018YFA0900300)the Tianjin Science Fund for Distinguished Young Scholars(17JCJQJC45300)the Science and Technology Service Network(STS)Initiative of the Chinese Academy of Sciences(CAS)(KFJ-STS-ZDTP-065).
文摘Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds.Here,the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli.First,glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001,resulting in the best candidate strain TS-1.Then icd and gdhA were overexpressed in TS-1,which led to the accumulation of 1.060 g/L tryptophan.Subsequently,one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-D-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine,resulting in the accumulation of 1.380 g/L tryptophan.Finally,to maintain cofactor balance,sthA and pntAB,encoding transhydrogenase,were overexpressed.With sufficient amounts of precursors and balanced cofactors,the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation,which was 2.76-times higher than the titer of the parent strain.Taken together,our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan.Similar strategies could be applied to the production of other amino acids or related derivatives.
基金This work was supported by the grants from National Key Research and Development Program of China(Grant number 2108YFA0107800)National Natural Science Foundation of China(Grant numbers 81920108004,81770107,81702722,81470362 and 81700168)+3 种基金National Postdoctoral Program for Innovative Talents(Grant number BX201700292)Natural Science Foundation of Hunan Province(Grant num ber 2018JJ3703)Science and Technology Key Project of Hunan Province(Grant number 2018SK21212)Fundamental Research Funds for the Central Universities of Central South University(Grant number 2018zzts386).
文摘Hematopoiesis requires finely tuned regulation of gene expression at each stage of development.The regulation of gene transcription involves not only individual transcription factors(TFs)but also transcription complexes(TCs)composed of transcription factor(s)and multisubunit cofactors.In their normal compositions,TCs orchestrate lineage-specific patterns of gene expression and ensure the production of the correct proportions of individual cell lineages during hematopoiesis.The integration of posttranslational and conformational modifcations in the chromatin landscape,nucleosomes,histones and interacting components via the cofactor-TF interplay is critical to optimal TF activity.Mutations or translocations of cofactor genes are expected to alter cofactor-TF interactions,which may be causative for the pathogenesis of various hematologic disorders.Blocking TF oncogenic activity in hematologic disorders through targeting cofactors in aberrant complexes has been an exciting therapeutic strategy.In this review,we summarize the current knowledge regarding the models and functions of cofactor-TF interplay in physiological hematopoiesis and highlight their implications in the etiology of hematological malignancies.This review presents a deep insight into the physiological and pathological implications of transcription machinery in the blood system.
