Production of Glucaric Acid from a Synthetic Pathway in Recombinant
            <i>Escherichia coli</i>

Moon, Tae Seok; Yoon, Sang-Hwal; Lanza, Amanda M.; Roy-Mayhew, Joseph D.; Prather, Kristala L. J.

doi:10.1128/aem.01065-09

Cited by 35 publications

(72 citation statements)

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“…The conversion of glucuronate to glucarate is known to be the fastest heterologous reaction in the biosynthesis of glucarate (29). Correspondingly, we see robust biosensor activation when glucuronate is the starting material.…”

Section: Resultsmentioning

confidence: 63%

“…Glucarate is a US Department of Energy "top value added chemical" to produce from biomass, with applications as a renewable replacement for petrochemicals and as a building block for new ultrahydrophilic polymers (15). Several papers describe construction and optimization of the glucarate biosynthesis pathway (28)(29)(30). However, attaining high titers remains challenging because of the low stability and activity of myo-inositol oxygenase, making this pathway a prime target for optimization by directed evolution.…”

Section: Resultsmentioning

confidence: 99%

“…6A) (11,29). We anticipated that the glucarate biosensor would produce a fluorescent response proportional to the amount of glucarate produced within the cell.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

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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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“…Previous studies on the production of D-glucaric acid showed that the MIOX-13 catalyzed step is limiting and that MIOX activity is strongly influenced by the 14 concentration of myo-inositol, its substrate (Moon et al, 2009a). We previously 15 designed a simple scaffold that co-recruits Ino1 and MIOX in a 1:1 ratio and showed that 16 D-glucaric acid titers were improved by 200% (Dueber et al, 2009).…”

Section: The Effect Of Ino1 and Miox Co-localization On D-glucaric Acmentioning

confidence: 99%

“…Control reactions were established without myo-inositol to 18 account for background. Assays for Ino1 and Udh activity were also performed using 19 lysates as described previously (Moon et al, 2009a). 20 21…”

Section: Assay For Activity Of Miox Ino1 and Udhmentioning

confidence: 99%

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

Moon

Dueber

Shiue

et al. 2010

Metabolic Engineering

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1The field of metabolic engineering has the potential to produce a wide variety of 2 chemicals in both an inexpensive and ecologically-friendly manner. Heterologous 3 expression of novel combinations of enzymes promises to provide new or improved 4 synthetic routes towards a substantially increased diversity of small molecules. Recently, 5we constructed a synthetic pathway to produce D-glucaric acid, a molecule that has been 6 deemed a "top-value added chemical" from biomass, starting from glucose. Limiting 7 flux through the pathway is the second recombinant step, catalyzed by myo-inositol 8 oxygenase (MIOX), whose activity is strongly influenced by the concentration of the 9 myo-inositol substrate. To synthetically increase the effective concentration of myo-10 inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-11 localize all three pathway enzymes in a designable complex as previously described 12 (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number 13 of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of 14 increased numbers of MIOX-targeted domains was much less significant. We 15 determined that the scaffolds directly increased the specific MIOX activity and that 16 glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed 17 an approximately 5-fold improvement in product titers over the non-scaffolded control, 18 and a 50% improvement over the previously reported highest titers. These results further 19 validate the utility of these synthetic scaffolds as a tool for metabolic engineering. 20 21

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Integrated Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers

Yang¹,

Yu²

2013

Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers

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Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

Cited by 35 publications

References 0 publications

Genetically encoded sensors enable real-time observation of metabolite production

Genetically encoded sensors enable real-time observation of metabolite production

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

Integrated Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers

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