scholarly journals Flux through Citrate Synthase Limits the Growth of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

2002 ◽  
Vol 68 (3) ◽  
pp. 1071-1081 ◽  
Author(s):  
S. A. Underwood ◽  
M. L. Buszko ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Ethanol Production ◽  
Citrate Synthase ◽  
Corn Steep Liquor ◽  
Pyruvate Decarboxylase ◽  
Luria Broth ◽  
Mineral Salts ◽  
E Coli ◽  
Steep Liquor

ABSTRACT 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-Km 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.

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

2004 ◽  
Vol 70 (5) ◽  
pp. 2734-2740 ◽  
Author(s):  
S. A. Underwood ◽  
M. L. Buszko ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Citrate Synthase ◽  
Corn Steep Liquor ◽  
Cell Mass ◽  
Functional Expression ◽  
Growth Conditions ◽  
Volumetric Productivity ◽  
Anaerobic Growth ◽  
Mineral Salts

ABSTRACT 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 [OD550] 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 OD550 unit−1), trehalose (9.9 nmol ml−1 OD550 unit−1), and betaine (19.8 nmol ml−1 OD550 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.

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

Specific Ethanol Production Rate in Ethanologenic Escherichia coli Strain KO11 Is Limited by Pyruvate Decarboxylase

2008 ◽  
Vol 15 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Gerardo Huerta-Beristain ◽  
José Utrilla ◽  
Georgina Hernández-Chávez ◽  
Francisco Bolívar ◽  
Guillermo Gosset ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ethanol Production ◽  
Production Rate ◽  
Pyruvate Decarboxylase ◽  
Ethanol Production Rate

Escherichia coli as a platform microbial host for systems metabolic engineering

2021 ◽  
Author(s):  
Dongsoo Yang ◽  
Cindy Pricilia Surya Prabowo ◽  
Hyunmin Eun ◽  
Seon Young Park ◽  
In Jin Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Metabolic Engineering ◽  
Food Ingredients ◽  
E Coli ◽  
Renewable Biomass ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Systems Metabolic Engineering ◽  
Microbial Host

Abstract Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

scholarly journals Silver Antibacterial Synergism Activities with Eight Other Metal(loid)-Based Antimicrobials against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 853
Author(s):  
Ali Pormohammad ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Silver Nitrate ◽  
Zinc Sulfate ◽  
Nickel Sulfate ◽  
Luria Broth ◽  
Potassium Tellurite ◽  
Wound Fluid ◽  
E Coli

The present study surveys potential antibacterial synergism effects of silver nitrate with eight other metal or metalloid-based antimicrobials (MBAs), including silver nitrate, copper (II) sulfate, gallium (III) nitrate, nickel sulfate, hydrogen tetrachloroaurate (III) trihydrate (gold), aluminum sulfate, sodium selenite, potassium tellurite, and zinc sulfate. Bacteriostatic and bactericidal susceptibility testing explored antibacterial synergism potency of 5760 combinations of MBAs against three bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) in three different media. Silver nitrate in combination with potassium tellurite, zinc sulfate, and tetrachloroaurate trihydrate had remarkable bactericidal and bacteriostatic synergism effects. Synergism properties of MBAs decreased effective antibacterial concentrations remarkably and bacterial cell count decreased by 8.72 log10 colony-forming units (CFU)/mL in E. coli, 9.8 log10 CFU/mL in S. aureus, and 12.3 log10 CFU/mL in P. aeruginosa, compared to each MBA alone. Furthermore, most of the MBA combinations inhibited the recovery of bacteria; for instance, the combination of silver nitrate–tetrachloroaurate against P. aeruginosa inhibited the recovery of bacteria, while three-fold higher concentration of silver nitrate and two-fold higher concentration of tetrachloroaurate were required for inhibition of recovery when used individually. Overall, higher synergism was typically obtained in simulated wound fluid (SWF) rather than laboratory media. Unexpectedly, the combination of A silver nitrate–potassium tellurite had antagonistic bacteriostatic effects in Luria broth (LB) media for all three strains, while the combination of silver nitrate–potassium tellurite had the highest bacteriostatic and bactericidal synergism in SWF. Here, we identify the most effective antibacterial MBAs formulated against each of the Gram-positive and Gram-negative pathogen indicator strains.

MICROBIAL PENTOSANASES: II. SOME FACTORS AFFECTING THE PRODUCTION OF PENTOSANASES BY BACILLUS PUMILUS AND BACILLUS SUBTILIS

1956 ◽  
Vol 2 (1) ◽  
pp. 28-38 ◽  
Author(s):  
F. J. Simpson
Keyword(s):  
Yeast Extract ◽  
Wheat Bran ◽  
Soybean Meal ◽  
Corn Steep Liquor ◽  
Ammonium Phosphate ◽  
Water Soluble ◽  
High Rate ◽  
Mineral Salts ◽  
Ammonium Lactate ◽  
Steep Liquor

A number of carbohydrates and nitrogenous adjuncts were tested for their effect on the constitutive and adaptive pentosanases produced by Bacillus stibtilis and B. pumilus respectively in a medium containing biotin, ammonium phosphate, and other mineral salts. B. subtilis produced more enzyme with sulphite liquor than with any of the other carbohydrate sources tested. Next, in decreasing order of merit, were wheat bran, maltose, ribose, beet molasses, oat hulls, and pectin. Of the nitrogenous adjuncts tested, corn steep liquor, soybean meal, gelatin, gelysate, and ammonium lactate doubled the yield of enzyme whereas yeast extract, peptone, urea, and others were less effective. For B. pumilus the better carbohydrate sources, in decreasing order of merit, were wheat bran, water soluble pentosan of wheat flour, xylan, straw holo-cellulose, wheat straw, and sulphite liquor. Of the nitrogen sources, corn steep liquor was outstanding while casein, casitone, phytone, yeast extract, distillers' dried solubles, and soybean meal followed in decreasing order. A medium containing 6% wheat bran (20 mesh), 1% corn steep liquor neutralized with ammonia, 0.05% sodium chloride, and 0.05% calcium carbonate was devised for the production of pentosanase by B. pumilus. With this medium in shaken Erlenmeyer flasks, the enzyme was produced at a high rate between 12 and 40 hr.; thereafter the rate of production decreased. Maximum yields were obtained in 96 hr. A temperature of 26 °C. was more favorable for pentosanase production than higher temperatures.

Nonequilibrium Plasma Decontamination of Corn Steep Liquor for Ethanol Production: SO2 Removal and Disinfection

2016 ◽  
Vol 6 (3-4) ◽  
pp. 219-234 ◽  
Author(s):  
Anh Huynh ◽  
Thomas Li ◽  
Mykola Kovalenko ◽  
Ryan D. Robinson ◽  
Alexander A. Fridman ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Corn Steep Liquor ◽  
Nonequilibrium Plasma ◽  
So2 Removal ◽  
Steep Liquor

pH-mediated control of anti-aggregation activities of cyanobacterial and E. coli chaperonin GroELs

2020 ◽  
Author(s):  
Tahmina Akter ◽  
Hitoshi Nakamoto
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Citrate Synthase ◽  
Ph Dependence ◽  
Chaperone Activity ◽  
Ph Change ◽  
Aggregation Assay ◽  
E Coli ◽  
Aggregation Activity

Abstract In contrast to Escherichia coli, cyanobacteria have multiple GroELs, the bacterial homologues of chaperonin/Hsp60. We have shown that cyanobacterial GroELs are mutually distinct and different from E. coli GroEL with which the paradigm for chaperonin structure/function has been established. However, little is known about regulation of cyanobacterial GroELs. This study investigated effect of pH (varied from 7.0 to 8.5) on chaperone activity of GroEL1 and GroEL2 from the cyanobacterium Synechococcus elongatus PCC7942 and E. coli GroEL. GroEL1 and GroEL2 showed pH dependency in suppression of aggregation of heat-denatured malate dehydrogenase, lactate dehydrogenase and citrate synthase. They exhibited higher anti-aggregation activity at more alkaline pHs. Escherichia coli GroEL showed a similar pH-dependence in suppressing aggregation of heat-denatured lactate dehydrogenase. No pH dependence was observed in all the GroELs when urea-denatured lactate dehydrogenase was used for anti-aggregation assay, suggesting that the pH-dependence is related to some denatured structures. There was no significant influence of pH on the chaperone activity of all the GroELs to promote refolding of heat-denatured malate dehydrogenase. It is known that pH in cyanobacterial cytoplasm increases by one pH unit following a shift from darkness to light, suggesting that the pH-change modulates chaperone activity of cyanobacterial GroEL1 and GroEL2.

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