Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol

Boyarskiy, Sergey; López, Stephanie Davis; Kong, Niwen; Tullman‐Ercek, Danielle

doi:10.1016/j.ymben.2015.11.005

Cited by 47 publications

(45 citation statements)

References 33 publications

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“…These results suggested that OmpC-TMT overexpression rescued the growth of the butanologenic E. coli strains, improved host robustness and increased n-butanol productivity. Our results are consistent with previous reports [28, 30, 31]. …”

Section: Resultssupporting

confidence: 94%

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Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

et al. 2017

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BackgroundN-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.ResultsThe host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.ConclusionsThese results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0356-3) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 94%

“…Boyarskiy et al [31] reported increased n-butanol tolerance and production (approximately 35% increases) through transcriptional feedback regulation of efflux protein expression.…”

Section: Resultsmentioning

confidence: 99%

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

et al. 2017

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“…This will no doubt include, for example, the prospecting for as well as engineering of efficient aromatic efflux pumps. Meanwhile, as known trade‐offs exist with respect to the overexpression of membrane transporters, for which an optimal balance between function and burden has been reported, more sophisticated control strategies for transporter expression in response to aromatic production levels might play a key role towards circumventing this caveat . Such prospective applications, meanwhile, also further highlight the importance of developing aromatic biosensors and their potential utility for allowing cells to autonomously respond to their changing production environment.…”

Section: Future Outlookmentioning

confidence: 99%

“…Meanwhile, as known trade-offs exist with respect to the overexpression of membrane transporters, for which an optimal balance between function and burden has been reported, 187 more sophisticated control strategies for transporter expression in response to aromatic production levels might play a key role towards circumventing this caveat. 188,189 Such prospective applications, meanwhile, also further highlight the importance of developing aromatic biosensors and their potential utility for allowing cells to autonomously respond to their changing production environment. Further identification and engineering of alternative hosts with greater inherent aromatic tolerance will also be important to addressing product toxicity.…”

Section: Future Outlookmentioning

confidence: 99%

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

Machas

Kurgan

Jha

et al. 2018

J of Chemical Tech & Biotech

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Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with a wide range of industrial and commercial applications. Significant progress has been made to alternatively and sustainably produce many aromatics from renewable substrates using microbial biocatalysts. While the construction of both natural and non‐natural pathways has expanded the number and diversity of aromatic bioproducts, pathway modularization in both single‐ and multi‐strain systems continues to support the enhancement of key production metrics towards economically‐viable levels. Emerging tools for implementing more precise metabolic control (e.g. CRISPRi, sRNA) as well as the engineering of novel high‐throughput screening platforms utilizing in vivo aromatic biosensors, meanwhile, continue to facilitate further optimization of both pathways and hosts. While product toxicity persists as a key challenge limiting the production of many aromatics, various successful strategies have been demonstrated towards improving tolerance, including via membrane and efflux pump engineering as well as by exploiting alternative production hosts. Finally, as a further step towards sustainable and economical aromatic bioproduction, non‐model substrates including lignin‐derived compounds continue to emerge as viable feedstocks. This review highlights recent and notable achievements related to such efforts while offering future outlooks towards engineering microbial cell factories for aromatic production. © 2018 Society of Chemical Industry

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“…When a sensor for a specific metabolite is unavailable, native promoters that respond to a given stimulus have also been co‐opted as sensors (Dahl et al , ; Yuan & Ching, ). However, many native promoters integrate multiple signals, making them respond to alternative or unknown stimuli (Kang et al , ; Dahl et al , ; Boyarskiy et al , ; Rajkumar et al , ; preprint: Borkowski et al , ; Hoynes‐O'Connor et al , ; Kasey et al , ; Siu et al , ). One approach to address this is to put the operators for a transcription factor into the “clean” background of a constitutive promoter (Cox et al , ).…”

Section: Introductionmentioning

confidence: 99%

Dynamic control of endogenous metabolism with combinatorial logic circuits

Moser

Borujeni

Ghodasara

et al. 2018

Molecular Systems Biology

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Controlling gene expression during a bioprocess enables real‐time metabolic control, coordinated cellular responses, and staging order‐of‐operations. Achieving this with small molecule inducers is impractical at scale and dynamic circuits are difficult to design. Here, we show that the same set of sensors can be integrated by different combinatorial logic circuits to vary when genes are turned on and off during growth. Three Escherichia coli sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by‐product accumulation (acetate) are constructed and optimized. By integrating these sensors, logic circuits implement temporal control over an 18‐h period. The circuit outputs are used to regulate endogenous enzymes at the transcriptional and post‐translational level using CRISPRi and targeted proteolysis, respectively. As a demonstration, two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed (pta or poxB) and respond by turning off the corresponding gene. This work demonstrates how simple circuits can be implemented to enable customizable dynamic gene regulation.

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Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol

Cited by 47 publications

References 33 publications

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

Dynamic control of endogenous metabolism with combinatorial logic circuits

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