Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which great...Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which greatly limits its application in the large-scale synthesis of chiral amines.In this study,a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy.Furthermore,conservation analysis,homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites,and the mutants L52S and T143C were obtained.The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg,respectively.The enzyme activity of the latter was 188%and the Tm value was 2.55°Chigher than those of the original mutant,which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.展开更多
This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter des...This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.展开更多
As a versatile type of enzyme,carboxylic acid reductases(CAR)can not only reduce various carboxylic acids to aldehydes in cooperation with cofactors ATP and NADPH but also catalyze the synthesis of amides and esters i...As a versatile type of enzyme,carboxylic acid reductases(CAR)can not only reduce various carboxylic acids to aldehydes in cooperation with cofactors ATP and NADPH but also catalyze the synthesis of amides and esters in the absence of NADPH.Here,we report an intramolecular cyclization catalyzed by CAR only with the use of ATP to transform amino acids into diverse lactams,includingγ-/δ-/ε-lactams and chiral derivatives thereof.The observed wide substrate scope and selectivity enable potential applications to be implemented.Our results demonstrate that CAR-catalyzed lactamization is a promising approach for the synthesis of chiral lactam com-pounds under mild conditions,thereby enriching the toolbox for the biosynthesis of lactams as a viable alternative to purely chemical procedures.展开更多
Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overpr...Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.展开更多
基金supported by the Key Technology Research Plan Project of the Inner Mongolia Autonomous Region(2019GG302)the Program of the Key Laboratory of Carbohydrate Chemistry&Biotechnology,Ministry of Education,China(KLCCB-KF202103).
文摘Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which greatly limits its application in the large-scale synthesis of chiral amines.In this study,a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy.Furthermore,conservation analysis,homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites,and the mutants L52S and T143C were obtained.The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg,respectively.The enzyme activity of the latter was 188%and the Tm value was 2.55°Chigher than those of the original mutant,which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.
文摘This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.
基金the Natural Science Foundation of Henan Province(182300410053)the China Postdoctoral Science Foundation(2020M672308)+3 种基金Henan Postdoctoral Science Foundation(227462)Science Project(902019AA0140)the National Key Research and Development Program of China(2019YFA0905100)the National Natural Science Foundation of China(U2004143)。
基金supported by the National Key Research and Development Program of China(No.2019YFA0905100)the National Natural Science Foundation of China(No.31900909)+1 种基金the Natural Science Foundation Applying a system of Tianjin(No.21JCJQJC00110)and Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(No.TSBICIP-CXRC-009)Mr Qu also thanks financial support from Youth Innovation Promotion Association,CAS(No.2021175).
文摘As a versatile type of enzyme,carboxylic acid reductases(CAR)can not only reduce various carboxylic acids to aldehydes in cooperation with cofactors ATP and NADPH but also catalyze the synthesis of amides and esters in the absence of NADPH.Here,we report an intramolecular cyclization catalyzed by CAR only with the use of ATP to transform amino acids into diverse lactams,includingγ-/δ-/ε-lactams and chiral derivatives thereof.The observed wide substrate scope and selectivity enable potential applications to be implemented.Our results demonstrate that CAR-catalyzed lactamization is a promising approach for the synthesis of chiral lactam com-pounds under mild conditions,thereby enriching the toolbox for the biosynthesis of lactams as a viable alternative to purely chemical procedures.
基金This study was supported by the National Key Research and Development Program of China(grant nos.2021YFC2100600,2019YFA0905100 and 2018YFA0901600)the National Natural Science Foundation of China(grant nos.31872614,32022002,21977113)+1 种基金the Youth Scientists Innovation Promotion Association of CAS(2019090)to S.S.G.,Innovative Cross Team project of Chinese Academy of Sciences,CAS(grant no.JCTD-2019-06)Shandong Provincial Natural Science Foundation(Major Basic Research Projects)(grant no.ZR2019ZD18).
文摘Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.
