Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport

Shiue, Eric; Prather, Kristala L. J.

doi:10.1016/j.ymben.2013.12.002

Cited by 85 publications

(94 citation statements)

References 46 publications

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“…The lag time observed at the high glucuronic acid concentration is comparable to the lag time observed with glucaric acid, supporting the previous finding that the Udh enzyme acts on a fast time scale compared with the selection (27,28). A long lag time even at a high concentration of myo-inositol indicates that the MIOX enzyme is less efficient, as reported in previous work (29). Shown are mutations found at targeted genes (Bottom) and those at untargeted genes (Center).…”

Section: Resultssupporting

confidence: 88%

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Evolution-guided optimization of biosynthetic pathways

Raman

Rogers

Taylor

et al. 2014

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

257

247

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Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼10 9 cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36-and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Wholegenome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.evolution | metabolic engineering | synthetic biology | sensors | biosynthetic pathways

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Section: Resultssupporting

confidence: 88%

“…Efforts to increase glucaric acid production in E. coli have focused on colocalization of pathway enzymes (30) and improving MIOX solubility (29). To date, modifying endogenous E. coli pathways has not been explored.…”

Section: Resultsmentioning

confidence: 99%

Evolution-guided optimization of biosynthetic pathways

Raman

Rogers

Taylor

et al. 2014

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

257

247

View full text Add to dashboard Cite

show abstract

“…Correspondingly, we see robust biosensor activation when glucuronate is the starting material. The fluorescent response to myo-inositol addition is slow and corroborates the difficulty of using M. musculus MIOX in the catalysis of myo-inositol to glucuronate in E. coli (30). The lack of biosensor response to additional glucose may reflect the fact that glucarate biosynthesis is competing with glycolysis for glucose-6-phosphate.…”

Section: Resultsmentioning

confidence: 60%

“…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.

164

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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“…In addition to the continued development of established bioproducts, further growth and diversification amongst the renewable biochemicals market will occur most significantly through the engineering of novel microbial biocatalysts capable of synthesizing new and useful products as renewable replacements to conventional petrochemicals. Several recent examples illustrate how this approach has already been used to broaden the spectrum of chemical products that can be synthesized from renewable biomass, including 1,4-butanediol [4], iso-butanol [5], isoprene [6], and various organic acids [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Creating pathways towards aromatic building blocks and fine chemicals

Thompson

Machas

Nielsen

2015

Current Opinion in Biotechnology

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Aromatic compounds represent a broad class of chemicals with a range of industrial applications, all of which are conventionally derived from petroleum feedstocks. However, owing to a diversity of available pathway precursors along with natural and engineered enzyme 'parts', microbial cell factories can be engineered to create alternative, renewable routes to many of the same aromatic products. Drawing from the latest tools and strategies in metabolic engineering and synthetic biology, such efforts are becoming an increasingly systematic practice, while continued efforts promise to open new doors to an ever-expanding range and diversity of renewable chemical and material products. This short review will highlight recent and notable achievements related for the microbial production of aromatic chemicals.

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Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport

Cited by 85 publications

References 46 publications

Evolution-guided optimization of biosynthetic pathways

Evolution-guided optimization of biosynthetic pathways

Genetically encoded sensors enable real-time observation of metabolite production

Creating pathways towards aromatic building blocks and fine chemicals

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