Journal ArticleDOI
Engineering dynamic pathway regulation using stress-response promoters
Robert H. Dahl,Fuzhong Zhang,Jorge Alonso-Gutierrez,Jorge Alonso-Gutierrez,Edward E. K. Baidoo,Edward E. K. Baidoo,Tanveer S. Batth,Tanveer S. Batth,Alyssa M. Redding-Johanson,Alyssa M. Redding-Johanson,Christopher J. Petzold,Christopher J. Petzold,Aindrila Mukhopadhyay,Aindrila Mukhopadhyay,Taek Soon Lee,Taek Soon Lee,Paul D. Adams,Paul D. Adams,Paul D. Adams,Jay D. Keasling +19 more
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TLDR
This paper applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product.Abstract:
Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.read more
Citations
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Journal ArticleDOI
Engineering Cellular Metabolism
TL;DR: How new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation is discussed.
Journal ArticleDOI
Principles of genetic circuit design
TL;DR: In this article, a review describes new tools that aid the construction of genetic circuits and discusses the failure modes encountered when assembling circuits, quantify their impact on performance, and review mitigation efforts.
Principles of genetic circuit design
TL;DR: Better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.
Journal ArticleDOI
Improving fatty acids production by engineering dynamic pathway regulation and metabolic control
TL;DR: A genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli was reported, able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis.
Journal ArticleDOI
Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications
TL;DR: This work discusses cell physiological responses to metabolic burdens, as well as strategies to identify and resolve the carbon and energy burden problems, including metabolic balancing, enhancing respiration, dynamic regulatory systems, chromosomal engineering, decoupling cell growth with production phases, and co- utilization of nutrient resources.
References
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Journal ArticleDOI
Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity.
TL;DR: A novel method to clone terpene synthase genes is developed, which relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype in E. coli engineered to produce elevated levels of isopentenol.
Journal ArticleDOI
Synthetic promoter libraries--tuning of gene expression.
TL;DR: Two different methods for obtaining promoter libraries are described and compared, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter.
Journal ArticleDOI
GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli.
Florence Hommais,Evelyne Krin,Jean-Yves Coppée,Céline Lacroix,Edouard Yeramian,Antoine Danchin,Philippe Bertin +6 more
TL;DR: Structural analysis of this chromosomal region suggests that the promoters of the corresponding genes are preferentially denatured, supporting the existence of a fitness island for acid adaptation on the E. coli chromosome.
Journal ArticleDOI
Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis.
TL;DR: This work has developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors, and demonstrates product-dependent growth in E. coli using all three compounds.
Journal ArticleDOI
Targeted proteomics for metabolic pathway optimization: application to terpene production.
Alyssa M. Redding-Johanson,Tanveer S. Batth,Rossana Chan,Rachel Krupa,Heather L. Szmidt,Paul D. Adams,Jay D. Keasling,Taek Soon Lee,Aindrila Mukhopadhyay,Christopher J. Petzold +9 more
TL;DR: Targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene and two mevalonate pathway proteins were identified as potential bottlenecks.
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