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

Moon, Tae Seok; Dueber, John E.; Shiue, Eric; Prather, Kristala L. J.

doi:10.1016/j.ymben.2010.01.003

Cited by 267 publications

(178 citation statements)

References 44 publications

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“…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%

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: 99%

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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“…Protein scaffolding is one of the most successful strategies used to create supramolecular enzyme complexes (24,31). However, enzyme cascades involving multimeric enzymes are often difficult to assemble, as they tend to result in large and disordered structures because of the random cross-linking between enzymes and scaffolds (31).…”

Section: Engineered Supramolecular Enzyme Complexes Enhance H6p and F6pmentioning

confidence: 99%

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

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

104

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Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. methane | methylotophs | supramolcular | scaffold | substrate channeling

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“…Previous studies have rewired the naturally occurring Ste5 scaffold in the yeast MAPK cascade to redirect signals, to modify delays in signaling time, and to introduce ultra sensitivity (Park et al , 2003;Bashor et al , 2008). Here we have taken synthetic scaffold proteins, which were designed and optimized to increase flux through metabolic pathways (Dueber et al , 2009;Moon et al , 2010;Whitaker and Dueber, 2011), and built an entirely synthetic feedback circuit. The system is designed with multiple inducible promoters and most components can be tuned.…”

Section: Discussionmentioning

confidence: 99%

Design and implementation of a synthetic biomolecular concentration tracker

Hsiao

Santos

Whitaker

et al. 2013

Preprint

Self Cite

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As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we design, model, and implement a biomolecular concentration tracker, in which an output protein tracks the concentration of an input protein. Using synthetic scaffolds built from small, modular protein-protein interaction domains to colocalize a two-component system, the circuit design relies on a single negative feedback loop to modulate the production of the output protein. Using a combination of modeling and experimental work, we show that the circuit achieves real-time protein concentration tracking in Escherichia coli and that steady state outputs can be tuned.

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

Cited by 267 publications

References 44 publications

Genetically encoded sensors enable real-time observation of metabolite production

Genetically encoded sensors enable real-time observation of metabolite production

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Design and implementation of a synthetic biomolecular concentration tracker

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