Engineering an<i>Acinetobacter</i>regulon for biosensing and high-throughput enzyme screening in<i>E. coli</i>via flow cytometry

Jha, Ramesh K.; Kern, Theresa L.; Fox, David T.; Strauss, Charlie E. M.

doi:10.1093/nar/gku444

Cited by 64 publications

(80 citation statements)

References 26 publications

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“…In contrast, genetically encoded biosensors have enabled FACS to be used for inconspicuous compounds. These studies have shown that biosensor-based FACS can be successful with maximum dynamic ranges as low as 12-fold, indicating that even poorly operating biosensor systems enable multiplexed phenotype evaluation (34). Implementing FACS with our techniques for real-time monitoring of whole pathway biosynthesis will enable greater versatility for phenotype evaluation in metabolic engineering.…”

Section: Discussionmentioning

confidence: 95%

Genetically encoded sensors enable real-time observation of metabolite production

Rogers

Church

2016

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

167

114

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Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.biotechnology | directed evolution | biosensor | metabolic engineering | synthetic biology B iological production of valuable products such as pharmaceuticals or renewable chemicals holds the potential to transform the global economy. However, the rate at which bioengineers are able to engineer new living catalysts is hampered by an exceedingly slow design-build-test cycle. We describe a method to accelerate the design-build-test cycle for metabolic engineering by enabling the observation of product formation within microbes as it occurs.Biological production of a desired product is accomplished by guiding a low-cost starting material such as glucose through a series of intracellular enzymatic reactions, ultimately yielding a molecule of economic interest. The choice of culture conditions, the creation of enzyme variants, and the tuning of endogenous cellular metabolism create a vast universe of potential designs. Because of the complexity of biology, appropriate genetic designs and culture conditions are not known a priori. Even sophisticated modeling paradigms can result in very large design spaces (1, 2). Evaluating genetic designs or modulating process parameters to achieve a desired outcome is therefore a major bottleneck in the bioengineering design-build-test cycle.Current methods...

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Section: Discussionmentioning

confidence: 95%

Genetically encoded sensors enable real-time observation of metabolite production

Rogers

Church

2016

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

167

114

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“…Previously, Fox and coworkers showed that 34DHB could be generated in Escherichia coli , but the critical enzyme, dehydroshikimate dehydratase, is suboptimal in activity and stability. As a prelude to library‐based optimization for this enzyme we were motivated to create an in vivo , single‐cell, selectable, reporter for 34DHB that operates in same strain of E. coli . In this work, we rationally redesign a TF that has no nascent activity to 34DHB.…”

Section: Introductionmentioning

confidence: 99%

Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor

et al. 2015

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Structure-based rational mutagenesis for engineering protein functionality has been limited by the scarcity and difficulty of obtaining crystal structures of desired proteins. On the other hand, when high-throughput selection is possible, directed evolution-based approaches for gaining protein functionalities have been random and fortuitous with limited rationalization. We combine comparative modeling of dimer structures, ab initio loop reconstruction, and ligand docking to select positions for mutagenesis to create a library focused on the ligand-contacting residues. The rationally reduced library requirement enabled conservative control of the substitutions by oligonucleotide synthesis and bounding its size within practical transformation efficiencies (∼ 10(7) variants). This rational approach was successfully applied on an inducer-binding domain of an Acinetobacter transcription factor (TF), pobR, which shows high specificity for natural effector molecule, 4-hydroxy benzoate (4HB), but no native response to 3,4-dihydroxy benzoate (34DHB). Selection for mutants with high transcriptional induction by 34DHB was carried out at the single-cell level under flow cytometry (via green fluorescent protein expression under the control of pobR promoter). Critically, this selection protocol allows both selection for induction and rejection of constitutively active mutants. In addition to gain-of-function for 34DHB induction, the selected mutants also showed enhanced sensitivity and response for 4HB (native inducer) while no sensitivity was observed for a non-targeted but chemically similar molecule, 2-hydroxy benzoate (2HB). This is unique application of the Rosetta modeling protocols for library design to engineer a TF. Our approach extends applicability of the Rosetta redesign protocol into regimes without a priori precision structural information.

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“…To further confirm the antibacterial activity of silica‐supported AgNPs against E. coli , flow cytometry was used to quantitatively measure the bacterial growth . After incubation with PI and 20 s irradiation, approximately 99% of E. coli remained viable with increased PI signal and these bacteria were consequently referred to as “live”.…”

Section: Resultsmentioning

confidence: 99%

A surfactant‐free synthesis of the silica nanosphere‐supported ultrafine silver nanoparticles and their antibacterial effects

Shen

Shan

Lü

et al. 2019

J Chinese Chemical Soc

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In this study, a facile, efficient, and surfactant‐free method to synthesize silica nanosphere‐supported ultrafine silver nanoparticles (AgNPs) (~2.5 nm) was developed, and their antibacterial effects were investigated. In the synthesis process, the hydrolysis of 3‐mercaptopropyltrimethoxysilane was adopted to provide thiol groups and in situ reduce Ag+ to Ag0 for ultrafine AgNPs formation on the surface of the silica nanosphere. Electron microscopy characterization of the complex formed revealed that the ultrafine AgNPs were not agglomerated and grow without any surfactants because there were no excess electrons transported from the shell to reduce the silver ions to silver atoms. The antibacterial effects of the supported ultrafine AgNPs with the surfactant‐free surface were evaluated against the Escherichia coli even at very low dosage. After incubation with 20 μg/mL silica‐supported AgNPs up to 120 min, 99.7% of the E. coli were inactivated, according to the bacterial viability measured by flow cytometry.

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Engineering anAcinetobacterregulon for biosensing and high-throughput enzyme screening inE. colivia flow cytometry

Cited by 64 publications

References 26 publications

Genetically encoded sensors enable real-time observation of metabolite production

Genetically encoded sensors enable real-time observation of metabolite production

Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor

A surfactant‐free synthesis of the silica nanosphere‐supported ultrafine silver nanoparticles and their antibacterial effects

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