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
In this paper, we consider a class of left invariant Riemannian metrics on Sp(n),which is invariant under the adjoint action of the subgroup Sp(n-3) × Sp(1) × Sp(1) × Sp(1).Based on the related formulae...In this paper, we consider a class of left invariant Riemannian metrics on Sp(n),which is invariant under the adjoint action of the subgroup Sp(n-3) × Sp(1) × Sp(1) × Sp(1).Based on the related formulae in the literature, we show that the existence of Einstein metrics is equivalent to the existence of solutions of some homogeneous Einstein equations. Then we use a technique of the Gr?bner basis to get a sufficient condition for the existence, and show that this method will lead to new non-naturally reductive metrics.展开更多
Path integral technique is discussed using Hamilton Jacobi method. The Hamilton Jacobi function of non-natural Lagrangian is obtained using separation of variables method. This function makes an important role in path...Path integral technique is discussed using Hamilton Jacobi method. The Hamilton Jacobi function of non-natural Lagrangian is obtained using separation of variables method. This function makes an important role in path integral quantization. The path integral is obtained as integration over the canonical phase space coordinates, which contains the generalized coordinate q and the generalized momentum p. One illustrative example is considered to explain the application of our formalism.展开更多
This research considered the effect of non-natural aging on the microstructural characteristics and mechanical properties of as-cast aluminum 6063 alloys. The samples were developed through a sand casting process and ...This research considered the effect of non-natural aging on the microstructural characteristics and mechanical properties of as-cast aluminum 6063 alloys. The samples were developed through a sand casting process and machined into tensile and impact test samples before carrying out solution heat treatment at 550?C (0.83 T<sub>m</sub>) on two parts of the samples while retaining one part as the control. The two parts were further divided into sets denoted A and B and were aged at 180?C (0.27 T<sub>m</sub>) and 160?C (0.24 T<sub>m</sub>), respectively, for 12 hours. The results showed that sample A has the optimal yield strength and ultimate tensile strength of 192 and 206 MPa, respectively. Likewise, the sample gave the highest impact strength value of about 9.63 J/mm<sup>2</sup>. The observed results were supported by the optical micrograph, which revealed that the sample has evenly dispersed precipitates in its microstructure. This is deemed responsible for the observed increase in strength of the sample.展开更多
We prove that Sp(2k+l)admits at least two non-naturally reductive Einstein metrics which are Ad(Sp(k)×Sp(k)×Sp(l))-invariant ifk<1.It implies that every compact simple Lie group Sp(n)for n≥4 admits at le...We prove that Sp(2k+l)admits at least two non-naturally reductive Einstein metrics which are Ad(Sp(k)×Sp(k)×Sp(l))-invariant ifk<1.It implies that every compact simple Lie group Sp(n)for n≥4 admits at least 2[(n-1)/3]non-naturally reductive left-invariant Einstein metrics.展开更多
Biological nitrogen fixation(BNF)and photosynthetic carbon fixation underpin food production and climate mitigation,yet natural systems are constrained by oxygen sensitivity,high energy demand,and inefficient catalyst...Biological nitrogen fixation(BNF)and photosynthetic carbon fixation underpin food production and climate mitigation,yet natural systems are constrained by oxygen sensitivity,high energy demand,and inefficient catalysts.This review synthesizes advances that recast these processes as engineering targets and proposes a conceptual roadmap that bridges synthetic symbioses with the synthetic biology of enzymes and pathways.For BNF,progress spans cross-kingdom strategies—from refactoring nif gene sets and targeting nitrogenase assembly to eukaryotic organelles,to engineering plant-associated diazotrophs,rhizosphere control circuits,and emerging nodule-like microenvironments.For carbon assimilation,new-to-nature CO_(2)-fixation modules and photorespiratory bypasses illustrate how pathway redesign and alternative carboxylases can circumvent key Calvin–Benson–Bassham limitations,and expanding photosynthetic light capture offers additional leverage.Across these domains,we extract common design principles:(i)nitrogenase output is increasingly governed by carbon/energy supply and electron delivery as much as by oxygen protection;(ii)robust function requires compartment-aware enzyme–chassis coordination,substrate channeling,and dynamic regulation using sensors and control circuits;and(iii)scalable implementation may benefit from distributing metabolic labor across engineered consortia rather than forcing all functions into a single host.We discuss enabling technologies—including AI-guided protein design and directed evolution,cell-free prototyping,chassis toolkits,and materials/bioelectrochemical interfaces—that can accelerate design–build–test–learn cycles and reduce barriers to deployment.Together,these insights define a path toward integrated nitrogen and carbon fixation systems for low-emission agriculture and biomanufacturing.展开更多
Biocatalysis,which involves using enzymes to address synthetic challenges of significance to humans,has rapidly developed into a pivotal technology for chemical innovation.Over the past decade,there has been a notable...Biocatalysis,which involves using enzymes to address synthetic challenges of significance to humans,has rapidly developed into a pivotal technology for chemical innovation.Over the past decade,there has been a notable increase in the use of metalloproteins as catalysts for abiotic,synthetically valuable carbene and nitrene transfer reactions.This trend highlights the adaptability of protein-based catalysts and our growing ability to harness this potential for novel enzyme chemistry.This review focuses on the most recent advancements in metalloenzyme-catalyzed carbene and nitrene transfer reactions,including cyclopropanation,carbene Y–H and C–H insertions,Doyle-Kirmse reactions,aldehyde olefinations,nitrene azide-to-aldehyde conversions,and nitrene C–H insertion.A variety of protein scaffolds have been engineered to offer varied levels of reactivity and selectivity towards pharmaceutically relevant compounds.The application of these new catalysts in preparative-scale synthesis underscores their emerging biotechnological significance.Furthermore,insights into key intermediate and determining factors in stereochemistry are offering valuable guidance for engineering metalloproteins,thereby expanding the scope and utility of these non-natural activities.展开更多
Previous studies have demonstrated that associative learning and experience play important roles in the string-pulling of bumblebees(Bombus terrestris).However,the features of the target(artificial flower with sugar r...Previous studies have demonstrated that associative learning and experience play important roles in the string-pulling of bumblebees(Bombus terrestris).However,the features of the target(artificial flower with sugar reward)and the string that bees learn in such tasks remain unknown.This study aimed to explore the specific aspects of the string-flower arrangement that bumblebees learn and how they prioritize these features.We show that bumblebees trained with string-pulling are sensitive to the flower stimuli;they exhibit a preference for pulling strings connected to flowers over strings that are not attached to a target.Additionally,they chose to pull strings attached to flowers of the same color and shape as experienced during training.The string feature also plays a crucial role for bumblebees when the flower features are identical.Furthermore,bees prioritized the features of the strings rather than the flowers when both cues were in conflict.Our results show that bumblebees solve string-pulling tasks by acquiring knowledge about the characteristics of both targets and strings,and contribute to a deeper understanding of the cognitive processes employed by bees when tackling non-natural skills.展开更多
We study the law of the iterated logarithm (LIL) for the maximum likelihood estimation of the parameters (as a convex optimization problem) in the generalized linear models with independent or weakly dependent (ρ-mix...We study the law of the iterated logarithm (LIL) for the maximum likelihood estimation of the parameters (as a convex optimization problem) in the generalized linear models with independent or weakly dependent (ρ-mixing) responses under mild conditions. The LIL is useful to derive the asymptotic bounds for the discrepancy between the empirical process of the log-likelihood function and the true log-likelihood. The strong consistency of some penalized likelihood-based model selection criteria can be shown as an application of the LIL. Under some regularity conditions, the model selection criterion will be helpful to select the simplest correct model almost surely when the penalty term increases with the model dimension, and the penalty term has an order higher than O(log log n) but lower than O(n). Simulation studies are implemented to verify the selection consistency of Bayesian information criterion.展开更多
Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be ...Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be engineered to remove its immunodominant N-acetylglucosamine(GlcNAc)side chain,implicated in provoking autoimmune cross-reactivity in rheumatic heart disease,leaving its polyrhamnose core(GACPR).Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization.Instead of conjugation to a standard carrier protein,we selected SpyAD,a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen.SpyAD was synthesized using the XpressTM cell-free protein expression system,incorporating a non-natural amino acid to which GACpr was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes.The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils.Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR,together with candidate vaccine antigens streptolysin O and C5a peptidase,protected against GAS challenge in a systemic infection model and localized skin infection model,without evidence of cross reactivity to human heart or brain tissue epitopes.This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.展开更多
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.
基金supported by NSFC (12071228,11901300, 51535008)Natural Science Research of Jiangsu Education Institutions of China (19KJB110015)。
文摘In this paper, we consider a class of left invariant Riemannian metrics on Sp(n),which is invariant under the adjoint action of the subgroup Sp(n-3) × Sp(1) × Sp(1) × Sp(1).Based on the related formulae in the literature, we show that the existence of Einstein metrics is equivalent to the existence of solutions of some homogeneous Einstein equations. Then we use a technique of the Gr?bner basis to get a sufficient condition for the existence, and show that this method will lead to new non-naturally reductive metrics.
文摘Path integral technique is discussed using Hamilton Jacobi method. The Hamilton Jacobi function of non-natural Lagrangian is obtained using separation of variables method. This function makes an important role in path integral quantization. The path integral is obtained as integration over the canonical phase space coordinates, which contains the generalized coordinate q and the generalized momentum p. One illustrative example is considered to explain the application of our formalism.
文摘This research considered the effect of non-natural aging on the microstructural characteristics and mechanical properties of as-cast aluminum 6063 alloys. The samples were developed through a sand casting process and machined into tensile and impact test samples before carrying out solution heat treatment at 550?C (0.83 T<sub>m</sub>) on two parts of the samples while retaining one part as the control. The two parts were further divided into sets denoted A and B and were aged at 180?C (0.27 T<sub>m</sub>) and 160?C (0.24 T<sub>m</sub>), respectively, for 12 hours. The results showed that sample A has the optimal yield strength and ultimate tensile strength of 192 and 206 MPa, respectively. Likewise, the sample gave the highest impact strength value of about 9.63 J/mm<sup>2</sup>. The observed results were supported by the optical micrograph, which revealed that the sample has evenly dispersed precipitates in its microstructure. This is deemed responsible for the observed increase in strength of the sample.
基金This work was supported in part by the National Natural Science Foundation of China(Nos.11571182,11901300,11931009)the Natural Science Foundation of Tianjin(No.19JCYBJC 30600)the Natural Science Research of Jiangsu Education Institutions of China(No.19K JB 110015).
文摘We prove that Sp(2k+l)admits at least two non-naturally reductive Einstein metrics which are Ad(Sp(k)×Sp(k)×Sp(l))-invariant ifk<1.It implies that every compact simple Lie group Sp(n)for n≥4 admits at least 2[(n-1)/3]non-naturally reductive left-invariant Einstein metrics.
基金supported by the funds of the Ministry of Science and Technology of China(2019YFA0904700)the National Natural Science Foundation of China(32471477)to Cheng Qi.
文摘Biological nitrogen fixation(BNF)and photosynthetic carbon fixation underpin food production and climate mitigation,yet natural systems are constrained by oxygen sensitivity,high energy demand,and inefficient catalysts.This review synthesizes advances that recast these processes as engineering targets and proposes a conceptual roadmap that bridges synthetic symbioses with the synthetic biology of enzymes and pathways.For BNF,progress spans cross-kingdom strategies—from refactoring nif gene sets and targeting nitrogenase assembly to eukaryotic organelles,to engineering plant-associated diazotrophs,rhizosphere control circuits,and emerging nodule-like microenvironments.For carbon assimilation,new-to-nature CO_(2)-fixation modules and photorespiratory bypasses illustrate how pathway redesign and alternative carboxylases can circumvent key Calvin–Benson–Bassham limitations,and expanding photosynthetic light capture offers additional leverage.Across these domains,we extract common design principles:(i)nitrogenase output is increasingly governed by carbon/energy supply and electron delivery as much as by oxygen protection;(ii)robust function requires compartment-aware enzyme–chassis coordination,substrate channeling,and dynamic regulation using sensors and control circuits;and(iii)scalable implementation may benefit from distributing metabolic labor across engineered consortia rather than forcing all functions into a single host.We discuss enabling technologies—including AI-guided protein design and directed evolution,cell-free prototyping,chassis toolkits,and materials/bioelectrochemical interfaces—that can accelerate design–build–test–learn cycles and reduce barriers to deployment.Together,these insights define a path toward integrated nitrogen and carbon fixation systems for low-emission agriculture and biomanufacturing.
文摘Biocatalysis,which involves using enzymes to address synthetic challenges of significance to humans,has rapidly developed into a pivotal technology for chemical innovation.Over the past decade,there has been a notable increase in the use of metalloproteins as catalysts for abiotic,synthetically valuable carbene and nitrene transfer reactions.This trend highlights the adaptability of protein-based catalysts and our growing ability to harness this potential for novel enzyme chemistry.This review focuses on the most recent advancements in metalloenzyme-catalyzed carbene and nitrene transfer reactions,including cyclopropanation,carbene Y–H and C–H insertions,Doyle-Kirmse reactions,aldehyde olefinations,nitrene azide-to-aldehyde conversions,and nitrene C–H insertion.A variety of protein scaffolds have been engineered to offer varied levels of reactivity and selectivity towards pharmaceutically relevant compounds.The application of these new catalysts in preparative-scale synthesis underscores their emerging biotechnological significance.Furthermore,insights into key intermediate and determining factors in stereochemistry are offering valuable guidance for engineering metalloproteins,thereby expanding the scope and utility of these non-natural activities.
基金funded by Fundamental Research Funds for the Central Universities(grant no.BLX202273)National Natural Science Foundation of China(grant nos.32301292 and 32271888).
文摘Previous studies have demonstrated that associative learning and experience play important roles in the string-pulling of bumblebees(Bombus terrestris).However,the features of the target(artificial flower with sugar reward)and the string that bees learn in such tasks remain unknown.This study aimed to explore the specific aspects of the string-flower arrangement that bumblebees learn and how they prioritize these features.We show that bumblebees trained with string-pulling are sensitive to the flower stimuli;they exhibit a preference for pulling strings connected to flowers over strings that are not attached to a target.Additionally,they chose to pull strings attached to flowers of the same color and shape as experienced during training.The string feature also plays a crucial role for bumblebees when the flower features are identical.Furthermore,bees prioritized the features of the strings rather than the flowers when both cues were in conflict.Our results show that bumblebees solve string-pulling tasks by acquiring knowledge about the characteristics of both targets and strings,and contribute to a deeper understanding of the cognitive processes employed by bees when tackling non-natural skills.
文摘We study the law of the iterated logarithm (LIL) for the maximum likelihood estimation of the parameters (as a convex optimization problem) in the generalized linear models with independent or weakly dependent (ρ-mixing) responses under mild conditions. The LIL is useful to derive the asymptotic bounds for the discrepancy between the empirical process of the log-likelihood function and the true log-likelihood. The strong consistency of some penalized likelihood-based model selection criteria can be shown as an application of the LIL. Under some regularity conditions, the model selection criterion will be helpful to select the simplest correct model almost surely when the penalty term increases with the model dimension, and the penalty term has an order higher than O(log log n) but lower than O(n). Simulation studies are implemented to verify the selection consistency of Bayesian information criterion.
基金supported by the CARB-X,Vaxcyte,Inc.,and NIH Grant(AI077780)NJG was supported in part by a Ruth L.Kirschstein Institutional National Research Award from the National Institute for General Medical Sciences(T32 GM008666)。
文摘Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be engineered to remove its immunodominant N-acetylglucosamine(GlcNAc)side chain,implicated in provoking autoimmune cross-reactivity in rheumatic heart disease,leaving its polyrhamnose core(GACPR).Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization.Instead of conjugation to a standard carrier protein,we selected SpyAD,a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen.SpyAD was synthesized using the XpressTM cell-free protein expression system,incorporating a non-natural amino acid to which GACpr was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes.The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils.Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR,together with candidate vaccine antigens streptolysin O and C5a peptidase,protected against GAS challenge in a systemic infection model and localized skin infection model,without evidence of cross reactivity to human heart or brain tissue epitopes.This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.
