Benzene‐Free Synthesis of Adipic Acid

Niu, Wei; Draths, K. M.; Frost, J. W.

doi:10.1021/bp010179x

Cited by 305 publications

(237 citation statements)

References 51 publications

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“…Production methods using renewable resources, such as lignocellulose, are potentially attractive, provided that they can be cost-effective. However, despite progress in microbial MA production, previous studies have suffered from low yields and titers and high accumulation of intermediate metabolites (17)(18)(19)(20)(21)(22). …”

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confidence: 99%

Engineering Escherichia coli coculture systems for the production of biochemical products

Zhang

Pereira

et al. 2015

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

269

199

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Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.metabolic engineering | microbial coculture | muconic acid | 4-hydroxybenzoic acidM etabolic engineering and synthetic biology have made great strides in constructing and optimizing metabolic pathways for biochemical product synthesis in a pure culture (1, 2). There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3, 4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5, 6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems. Different from previous modular engineering approaches, such coculturebased systems completely modularize and segregate a biosynthetic pathway into two separate cells, each of which carries a portion of the pathway, and thus can be engineered independently to achieve optimal functioning of the combined pathway.The concept of microbial cocultures is not new. However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7-11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12, 13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency s...

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confidence: 99%

Engineering Escherichia coli coculture systems for the production of biochemical products

Zhang

Pereira

et al. 2015

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

269

199

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“…In this work, we develop a strategy for real-time monitoring of metabolic product formation and demonstrate its utility in observing the production of 3-hydroxypropionate (3HP; a renewable plastic precursor) (14), acrylate (the monomer for several common plastics), glucarate (a renewable building block for superabsorbent polymers and a replacement for phosphates in detergents) (15), and muconate (a building block for renewable nylon) (16). We develop two unique biosensors for 3HP and compare their ability to observe 3HP production.…”

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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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“…The sugar constituents of lignocellulose can be effectively metabolized by Escherichia coli, a commercially important biocatalyst for the production of amino acids and recombinant proteins (1,11). This organism has recently been engineered for the production of a variety of bulk chemicals, such as organic acids (4,8,18,20,36,47,53), diols (17,35,44), and fuel ethanol (22). Ethanologenic E. coli KO11 was previously constructed by the chromosomal integration of Zymomonas mobilis genes encoding a pyruvate decarboxylase with a low K m (pdc) and alcohol dehydrogenase II (adhB) (37).…”

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confidence: 99%

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2004

Appl Environ Microbiol

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Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml ؊1 optical density at 550 nm [OD 550 ] unit ؊1 ), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml ؊1 OD 550 unit ؊1 ), trehalose (9.9 nmol ml ؊1 OD 550 unit ؊1 ), and betaine (19.8 nmol ml ؊1 OD 550 unit ؊1 ). Two independent genetic modifications of E. coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.

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Benzene‐Free Synthesis of Adipic Acid

Cited by 305 publications

References 51 publications

Engineering Escherichia coli coculture systems for the production of biochemical products

Engineering Escherichia coli coculture systems for the production of biochemical products

Genetically encoded sensors enable real-time observation of metabolite production

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

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