In synthetic biology,researchers assemble biological components in new ways to produce systems with practical applications.One of these practical applications is control of the flow of genetic information(from nucleic...In synthetic biology,researchers assemble biological components in new ways to produce systems with practical applications.One of these practical applications is control of the flow of genetic information(from nucleic acid to protein),a.k.a.gene regulation.Regulation is critical for optimizing protein(and therefore activity)levels and the subsequent levels of metabolites and other cellular properties.The central dogma of molecular biology posits that information flow commences with transcription,and accordingly,regulatory tools targeting transcription have received the most attention in synthetic biology.In this mini-review,we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription.In particular,we focus on engineering studies where promoters and transcription terminators(cis-factors)were directly engineered and/or isolated from DNA libraries.We also review several well-characterized transcription regulators(trans-factors),giving examples of how cis-and trans-acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals.Last,we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits.While most of our mini-review focuses on the well-characterized bacterium Escherichia coli,we also provide several examples of the use of transcription control engineering in non-model organisms.Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.展开更多
Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-...Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing strains.We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced(NADPH-dependent)or redox-balanced(NADH-dependent)ketol-acid reductoisomerase enzyme.We then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains.Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes.Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version,albeit at low overall pathway flux.Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters,which are required cofactors for the dihydroxyacid dehydratase enzyme.We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron levels.The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold,demonstrating that both localizations can support flux to isobutanol.展开更多
基金MDE was supported by an NHGRI training grant to the Genomic Sciences Training Program 5T32-HG002760BFP was supported by a grant from the National Science Foundation(EFRI-1240268).
文摘In synthetic biology,researchers assemble biological components in new ways to produce systems with practical applications.One of these practical applications is control of the flow of genetic information(from nucleic acid to protein),a.k.a.gene regulation.Regulation is critical for optimizing protein(and therefore activity)levels and the subsequent levels of metabolites and other cellular properties.The central dogma of molecular biology posits that information flow commences with transcription,and accordingly,regulatory tools targeting transcription have received the most attention in synthetic biology.In this mini-review,we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription.In particular,we focus on engineering studies where promoters and transcription terminators(cis-factors)were directly engineered and/or isolated from DNA libraries.We also review several well-characterized transcription regulators(trans-factors),giving examples of how cis-and trans-acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals.Last,we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits.While most of our mini-review focuses on the well-characterized bacterium Escherichia coli,we also provide several examples of the use of transcription control engineering in non-model organisms.Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.
文摘Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing strains.We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced(NADPH-dependent)or redox-balanced(NADH-dependent)ketol-acid reductoisomerase enzyme.We then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains.Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes.Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version,albeit at low overall pathway flux.Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters,which are required cofactors for the dihydroxyacid dehydratase enzyme.We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron levels.The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold,demonstrating that both localizations can support flux to isobutanol.
