ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in <i>E. coli</i>

Xu, Peng; Vansiri, Amérin; Bhan, Namita; Koffas, Mattheos

doi:10.1021/sb300016b

Cited by 235 publications

(177 citation statements)

References 55 publications

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“…2a). Using a recently developed synthetic biology platform that streamlines the process of gene assembly and pathway construction 26 , the GLY, ACA and FAS modules were successfully expressed on the five compatible ePathBrick vectors (Supplementary Table S1), with varying promoter strength, plasmid copy number and antibiotic resistance marker.…”

Section: Resultsmentioning

confidence: 99%

“…NdeI/XhoI-digested PCR fragments were ligated to the NdeI and XhoI-digested pETM6 to give constructs pETM6-fabA, pETM6-fabG, pETM6-fabI and pETM6-tesA 0 , respectively. ePathBrick-directed gene assembly was performed based on these individual constructs 26 . Briefly, pseudo-operon configuration was achieved by ligating the AvrII/SalI-digested donor construct with the SpeI/SalI-digested receiver construct.…”

Section: Discussionmentioning

confidence: 99%

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Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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“…To construct FAS pathway, E. coli tesA′, fabA, fabD, fabG, and fabI gene fragments were assembled into artificial operon form based on the ePathBrick gene assembly platform (48). Briefly, the XbaI/SalI-digested donor vector was ligated to the SpeI/SalI-digested recipient vector to give an operon gene configuration.…”

Section: Methodsmentioning

confidence: 99%

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7-and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA-derived compounds.biofuels | dynamic metabolic control | transcriptional regulation A grand challenge in synthetic biology is to move the design of biomolecular circuits from purely genetic constructs toward systems that integrate different levels of cellular complexity, including regulatory networks and metabolic pathways (1). Despite the fact that a large volume of regulatory architectures and motifs has been discovered (2, 3), little has been accomplished in pathway engineering to improve cellular productivity and yield by exploiting dynamic pathway regulation and metabolic control (4). One essential part in implementing synthetic metabolic control in pathway engineering is to engineer novel metabolite sensors with desired input-output relationships. For example, have designed and applied a regulatory circuit that can sense the glycolytic pathway hallmark metabolite acetylphosphate to control the lycopene biosynthetic pathway (5) and generate oscillatory gene expression (6) as well as achieve artificial cell-cell communication (7). Dahl et al. (8) have used stressresponse promoters to improve farnesyl pyrophosphate production, and Tsao et al. (9) have rewired the Escherichia coli native quorumsensing regulon for autonomous induction of recombinant proteins.Traditional metabolic engineering is largely focused on the overexpression of rate-limiting steps (10

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“…Moreover, the large amount of data generated over the last decades allowed the construction of genome-scale metabolic models (3,4) leading to this organism being recognized as the 'green' Escherichia coli (5). Nevertheless, the vast majority of the available synthetic biology tools have been developed for heterotrophic chassis like E. coli or Saccharomyces cerevisiae (6,7), and most of the regulatory elements characterized in E. coli function rather poorly or not at all in cyanobacteria (8,9). Consequently, a considerable effort has been placed on the design and construction of efficient, predictable and easy-to-use molecular tools for cyanobacteria (8,(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters

et al. 2018

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Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO 2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

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ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli

Cited by 235 publications

References 55 publications

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters

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