The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit compo...The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components.Previously,extracytoplasmic function sigma factors(ECFs),a diverse group of alternative sigma factors with distinct promoter specificities,were shown to have great potential as context-independent regulators,but so far,they have only been used in a few model species.Here,we show that the alphaproteobacterium Sinorhizobium meliloti,which has been proposed as a plant-associated bacterial chassis for synthetic biology,has a similar phylogenetic ECF acceptance range as the gammaproteobacterium Escherichia coli.A common set of orthogonal ECF-based regulators that can be used in both bacterial hosts was identified and used to create 2-step delay circuits.The genetic circuits were implemented in single copy in E.coli by chromosomal integration using an established method that utilizes bacteriophage integrases.In S.meliloti,we demonstrated the usability of single-copy pABC plasmids as equivalent carriers of the synthetic circuits.The circuits were either implemented on a single pABC or modularly distributed on 3 such plasmids.In addition,we provide a toolbox containing pABC plasmids compatible with the Golden Gate(MoClo)cloning standard and a library of basic parts that enable the construction of ECF-based circuits in S.meliloti and in E.coli.This work contributes to building a context-independent and species-overarching ECF-based toolbox for synthetic biology applications.展开更多
Seven new polyketides,termed veramycins,were isolated from a Streptomyces sp.from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123.Veramycin A,anα-pyrone congener of TM-123 ...Seven new polyketides,termed veramycins,were isolated from a Streptomyces sp.from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123.Veramycin A,anα-pyrone congener of TM-123 and NFAT-133 showed an increased baseline deoxy-glucose uptake in the absence of insulin in a modified L6 rat skeletal muscle cell line(L6 GLUT4 AS160-like cells).In addition,both compounds slightly increased the sensitivity to insulin in this cell line.Total syntheses of NFAT-133,TM-123 and veramycin A were accomplished starting from a central building block,which bears the three contiguous stereogenic centers of this polyketide family.Our approach enables an efficient,selective and flexible access to all possible isomers of the stereotriad for further exploration of this series as a potential anti-diabetic lead structure as exemplified by the synthesis of an NFAT-133 epimer.Finally,the corresponding biosynthetic gene cluster(BGC)was identified by genome sequencing and gene inactivation.Based on feeding experiments,a biosynthetic pathway was proposed,which enabled access to new veramycin A analogs by precursor-directed biosynthesis.展开更多
基金supported by the ERASynBio program of the Federal Ministry of Education and Research(Germany,grant 031L0010B)the LOEWE program of the State of Hesse(Germany)through funding of the Center for Synthetic Microbiology(SYNMIKRO).
文摘The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components.Previously,extracytoplasmic function sigma factors(ECFs),a diverse group of alternative sigma factors with distinct promoter specificities,were shown to have great potential as context-independent regulators,but so far,they have only been used in a few model species.Here,we show that the alphaproteobacterium Sinorhizobium meliloti,which has been proposed as a plant-associated bacterial chassis for synthetic biology,has a similar phylogenetic ECF acceptance range as the gammaproteobacterium Escherichia coli.A common set of orthogonal ECF-based regulators that can be used in both bacterial hosts was identified and used to create 2-step delay circuits.The genetic circuits were implemented in single copy in E.coli by chromosomal integration using an established method that utilizes bacteriophage integrases.In S.meliloti,we demonstrated the usability of single-copy pABC plasmids as equivalent carriers of the synthetic circuits.The circuits were either implemented on a single pABC or modularly distributed on 3 such plasmids.In addition,we provide a toolbox containing pABC plasmids compatible with the Golden Gate(MoClo)cloning standard and a library of basic parts that enable the construction of ECF-based circuits in S.meliloti and in E.coli.This work contributes to building a context-independent and species-overarching ECF-based toolbox for synthetic biology applications.
文摘Seven new polyketides,termed veramycins,were isolated from a Streptomyces sp.from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123.Veramycin A,anα-pyrone congener of TM-123 and NFAT-133 showed an increased baseline deoxy-glucose uptake in the absence of insulin in a modified L6 rat skeletal muscle cell line(L6 GLUT4 AS160-like cells).In addition,both compounds slightly increased the sensitivity to insulin in this cell line.Total syntheses of NFAT-133,TM-123 and veramycin A were accomplished starting from a central building block,which bears the three contiguous stereogenic centers of this polyketide family.Our approach enables an efficient,selective and flexible access to all possible isomers of the stereotriad for further exploration of this series as a potential anti-diabetic lead structure as exemplified by the synthesis of an NFAT-133 epimer.Finally,the corresponding biosynthetic gene cluster(BGC)was identified by genome sequencing and gene inactivation.Based on feeding experiments,a biosynthetic pathway was proposed,which enabled access to new veramycin A analogs by precursor-directed biosynthesis.
