The commercial production of chemicals using pathway engineering

Chotani, Gopal; Dodge, Tim; Hsu, Amy P.; Kumar, Manoj; LaDuca, R J; Trimbur, D.; Weyler, Walter; Sanford, Karl

doi:10.1016/s0167-4838(00)00234-x

Cited by 210 publications

(102 citation statements)

References 83 publications

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“…One such widely used strategy is the high-efficiency PET cassette derived from Z. mobilis [14]. For success in the production of alternative biofuels, similar metabolic engineering strategies will be necessary; for example, the development of targeted and efficient transport systems, the improvement of the resistance of biofuels producers to the toxic effects of accumulating biomolecules, the optimization of carbon flux to the desired products, and the construction of strains that are robust under industrial process conditions [14,[72][73][74]. Some advances that might have enormous potential in the field of microbial biofuel production include the engineer-ing of a reduced-genome E. coli strain that can be used for the systematic design of desired phenotypes [75], and the inter-microbial genome transplantation demonstrated in Mycoplasma caprilocum [76].…”

Section: Box 2 Microbial Sources Of Other Petrochemical Commoditiesmentioning

confidence: 99%

Biofuel alternatives to ethanol: pumping the microbial well

Fortman

Chhabra

Mukhopadhyay

et al. 2008

Trends in Biotechnology

338

209

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Section: Box 2 Microbial Sources Of Other Petrochemical Commoditiesmentioning

confidence: 99%

Biofuel alternatives to ethanol: pumping the microbial well

Fortman

Chhabra

Mukhopadhyay

et al. 2008

Trends in Biotechnology

338

209

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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).…”

mentioning

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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“…This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility.…”

mentioning

confidence: 99%

“…The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility.…”

mentioning

confidence: 99%

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD ؉ . Expression of low-K m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility. Undesirable changes, such as reduced growth, decreased glycolytic flux, and low volumetric productivity, are generally attributed to a lack of ATP (19, 50), creation of futile cycles (10,11,37), changes in intracellular metabolite pools, or a metabolic imbalance (2,7,9,13,28,51,53). Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are apparent only in more minimal media (7,10,11,31).Salmonella enterica serovar Typhimurium was engineered for succinate production by increasing expression of pyc encoding pyruvate carboxylase (50). Although succinate production increased, the growth rate declined by 18% and the glycolytic flux decreased by 40%. Similar results were reported for an analogous construction in Escherichia coli (19). Donnelly and cow...

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The commercial production of chemicals using pathway engineering

Cited by 210 publications

References 83 publications

Biofuel alternatives to ethanol: pumping the microbial well

Biofuel alternatives to ethanol: pumping the microbial well

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

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

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