l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum...l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum is an attractive chassis for l-threonine production.However,the present l-threonine production in C.glutamicum cannot meet the requirement of industrialization due to the relatively low production level of l-threonine and the accumulation of large amounts of by-products(such as l-lysine,l-isoleucine,and glycine).Herein,to enhance the l-threonine biosynthesis in C.glutamicum,releasing the aspartate kinase(LysC)and homoserine dehydrogenase(Hom)from feedback inhibition by l-lysine and l-threonine,respectively,and overexpressing four flux-control genes were performed.Next,to reduce the formation of by-products l-lysine and l-isoleucine without the cause of an auxotrophic phenotype,the feedback regulation of dihydrodipicolinate synthase(DapA)and threonine dehydratase(IlvA)was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by l-lysine and l-isoleucine,respectively.The resulting strain maintained the capability of synthesizing enough amounts of l-lysine and l-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids.To further enhance l-threonine production and reduce the by-product glycine,l-threonine exporter and homoserine kinase were overexpressed.Finally,the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L(17.2%higher)l-threonine with a productivity of 1.20 g/L/h(108.0%higher)in fed-batch fermentation,along with significantly reduced by-product accumulation,representing the record for l-threonine production in C.glutamicum.In this study,we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic l-threonine producing C.glutamicum.The main end by-products including l-lysine,l-isoleucine,and glycine were almost eliminated in fed-batch fermentation of the engineered C.glutamicum strain.This strategy can also be used for engineering producing strains for other amino acids and derivatives.展开更多
To effectively exploit the tumor microenvironment(TME),TME-responsive nanocarriers based on cascade reactions have received much attention.In this study,we designed a novel nanoparticle PB@SiO_(2)@MnO_(2)@P-Arg(PMP)to...To effectively exploit the tumor microenvironment(TME),TME-responsive nanocarriers based on cascade reactions have received much attention.In this study,we designed a novel nanoparticle PB@SiO_(2)@MnO_(2)@P-Arg(PMP)to construct a cascade reaction nanoplatform.While using biosafety Prussian blue(PB)for photothermal therapy(PTT),this nanoplatform uses silica(SiO_(2))as an intermediate layer to assemble Prussian blue and manganese dioxide(MnO_(2))into a core-shell structure,which effectively enhances the response of the nanoplatform to TME and promotes the effect of chemodynamic therapy(CDT)resulting from glutathione(GSH)depletion and Fenton-like reaction.The released Mn2t can also be used for magnetic resonance imaging(MRI).Through the cascade reaction,poly-L-arginine(PArg)coated on the surface of the nanoparticles can react with hydroxyl radical(·OH)obtained from the Fenton-like reaction to release nitric oxide(NO),which further reacts with O_(2)^(-)·to produce the more toxic peroxynitrite anion(ONOO^(-)).The photothermal effect of PB further enhances the effect of the cascade reaction while reducing the amount of heat required for treatment.In vitro and in vivo studies confirmed the antitumor effects of cascade reaction-based nanoplatforms in combined photothermal/chemodynamic/gas cancer therapies,providing new strategies for the design and fabrication of multifunctional nanoplatforms that integrate diagnostic and therapeutic functions,as well as the application of cascade reactions in multimodal synergistic therapy.展开更多
基金funded by the National Key Research and Development Program of China(2021YFC2100900)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-CXRC-058)+2 种基金the National Natural Science Foundation of China(32270101)the Key R&D Program of Shandong Province(2021CXGC010602)the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)。
文摘l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum is an attractive chassis for l-threonine production.However,the present l-threonine production in C.glutamicum cannot meet the requirement of industrialization due to the relatively low production level of l-threonine and the accumulation of large amounts of by-products(such as l-lysine,l-isoleucine,and glycine).Herein,to enhance the l-threonine biosynthesis in C.glutamicum,releasing the aspartate kinase(LysC)and homoserine dehydrogenase(Hom)from feedback inhibition by l-lysine and l-threonine,respectively,and overexpressing four flux-control genes were performed.Next,to reduce the formation of by-products l-lysine and l-isoleucine without the cause of an auxotrophic phenotype,the feedback regulation of dihydrodipicolinate synthase(DapA)and threonine dehydratase(IlvA)was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by l-lysine and l-isoleucine,respectively.The resulting strain maintained the capability of synthesizing enough amounts of l-lysine and l-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids.To further enhance l-threonine production and reduce the by-product glycine,l-threonine exporter and homoserine kinase were overexpressed.Finally,the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L(17.2%higher)l-threonine with a productivity of 1.20 g/L/h(108.0%higher)in fed-batch fermentation,along with significantly reduced by-product accumulation,representing the record for l-threonine production in C.glutamicum.In this study,we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic l-threonine producing C.glutamicum.The main end by-products including l-lysine,l-isoleucine,and glycine were almost eliminated in fed-batch fermentation of the engineered C.glutamicum strain.This strategy can also be used for engineering producing strains for other amino acids and derivatives.
基金supported by the National Natural Science Foundation of China(Grant Nos.:51973053 and 51773055).
文摘To effectively exploit the tumor microenvironment(TME),TME-responsive nanocarriers based on cascade reactions have received much attention.In this study,we designed a novel nanoparticle PB@SiO_(2)@MnO_(2)@P-Arg(PMP)to construct a cascade reaction nanoplatform.While using biosafety Prussian blue(PB)for photothermal therapy(PTT),this nanoplatform uses silica(SiO_(2))as an intermediate layer to assemble Prussian blue and manganese dioxide(MnO_(2))into a core-shell structure,which effectively enhances the response of the nanoplatform to TME and promotes the effect of chemodynamic therapy(CDT)resulting from glutathione(GSH)depletion and Fenton-like reaction.The released Mn2t can also be used for magnetic resonance imaging(MRI).Through the cascade reaction,poly-L-arginine(PArg)coated on the surface of the nanoparticles can react with hydroxyl radical(·OH)obtained from the Fenton-like reaction to release nitric oxide(NO),which further reacts with O_(2)^(-)·to produce the more toxic peroxynitrite anion(ONOO^(-)).The photothermal effect of PB further enhances the effect of the cascade reaction while reducing the amount of heat required for treatment.In vitro and in vivo studies confirmed the antitumor effects of cascade reaction-based nanoplatforms in combined photothermal/chemodynamic/gas cancer therapies,providing new strategies for the design and fabrication of multifunctional nanoplatforms that integrate diagnostic and therapeutic functions,as well as the application of cascade reactions in multimodal synergistic therapy.
