A Constraint-based modeling approach to reach an improved chemically defined minimal medium for recombinant antiEpEX-scFv production by Escherichia coli

Increasing demand for recombinant therapeutic proteins highlights the necessity of their yield improvement. Culture medium formulation is a popular approach for bioprocess optimization to improve therapeutic protein production. Constraint-based modeling can empower high-precision optimization through information on how media compounds affect metabolism and cell growth. In the current study, a genome-scale metabolic model (GEMM) of Escherichia coli cells was employed to design strategies of minimal medium supplementation for higher antiEpEX-scFv production. Dynamic flux balance analysis of the recombinant E. coli cell model predicted that ammonium was depleted during the process. Based on the simulations, three amino acids (Asn, Gln and Arg) were chosen to be added to the medium to compensate for ammonium depletion. Experimental validation suggested that the addition of these amino acids (one-by-one, or in combinations) can indeed improve cell growth and recombinant protein production. Then, design of experiment was used to optimize the concentrations of amino acids in the growth medium. About two-fold increase in the growth rate and total scFv expression level was observed using this strategy. We conclude that the GEMM-based approach can provide insights into an effective feeding strategy to improve the production of recombinant protein in E. coli.

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Modification by an R determinant for streptomycin of hissd phenotype in a mutant strain of Escherichia coli B

Genetics Research ◽

10.1017/s0016672300011721 ◽

1968 ◽

Vol 12 (2) ◽

pp. 109-116 ◽

Cited By ~ 1

Author(s):

A. M. Molina ◽

L. Calegari ◽

G. Conte

Keyword(s):

Escherichia Coli ◽

Cell Membrane ◽

Mutant Strain ◽

Minimal Medium ◽

Specific Requirement ◽

Resistance Level ◽

E Coli ◽

Level Characteristic ◽

Escherichia Coli B

When an R determinant for streptomycin is transferred into a conditionally streptomycin-dependent E. coli B mutant—which requires in minimal medium either histidine or streptomycin—the latter behaves like a histidineless strain. This phenotype modification shows that the repairing action of streptomycin is prevented. The specific requirement of the strain is not now replaced even by streptomycin concentrations up to 10000 µg/ml at which the conditionally streptomycin-dependent mutant could originally grow, and which are well beyond the resistance level characteristic of the R determinant itself. These data seem to suggest that a reduction in permeability of the cell membrane cannot be held responsible for the phenomenon observed.

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Effect of ascorbate on oxygen uptake and growth of Escherichia coli B

Canadian Journal of Microbiology ◽

10.1139/m88-140 ◽

1988 ◽

Vol 34 (6) ◽

pp. 822-824 ◽

Cited By ~ 9

Author(s):

Holly E. Richter ◽

Jacek Switala ◽

Peter C. Loewen

Keyword(s):

Escherichia Coli ◽

Oxygen Uptake ◽

Electron Acceptor ◽

Minimal Medium ◽

Anaerobic Growth ◽

Rich Medium ◽

Terminal Electron Acceptor ◽

Subsequent Change ◽

Escherichia Coli B

The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth. The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium. Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate. Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor. Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.

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Roles of the Extraintestinal Pathogenic Escherichia coli ZnuACB and ZupT Zinc Transporters during Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.01082-08 ◽

2008 ◽

Vol 77 (3) ◽

pp. 1155-1164 ◽

Cited By ~ 70

Author(s):

Mourad Sabri ◽

Sébastien Houle ◽

Charles M. Dozois

Keyword(s):

Escherichia Coli ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Minimal Medium ◽

Cumulative Effect ◽

Transport Systems ◽

Single Strain ◽

Pathogenic Escherichia Coli ◽

K 12

ABSTRACT Roles of the ZnuACB and ZupT transporters were assessed in Escherichia coli K-12 and uropathogenic E. coli (UPEC) CFT073. K-12 and CFT073 Δznu ΔzupT mutants demonstrated decreased 65Zn2+ uptake and growth in minimal medium. CFT073Δznu demonstrated an intermediate decrease of 65Zn2+ uptake and growth in minimal medium, whereas the CFT073ΔzupT mutant grew as well as CFT073 and exhibited a less marked decrease in 65Zn2+ uptake. CFT073 mutants grew as well as the wild type in human urine. In competitive infections in CBA/J mice, the ΔzupT mutant demonstrated no disadvantage during urinary tract infection. In contrast, the UPEC Δznu and Δznu ΔzupT strains demonstrated significantly reduced numbers in the bladders (mean 4.4- and 30-fold reductions, respectively) and kidneys (mean 41- and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the Δznu and Δznu ΔzupT mutants were reduced in the kidneys (P = 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 Δznu ΔzupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.

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The Putrescine Importer PuuP of Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.01314-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2776-2782 ◽

Cited By ~ 36

Author(s):

Shin Kurihara ◽

Yuichi Tsuboi ◽

Shinpei Oda ◽

Hyeon Guk Kim ◽

Hidehiko Kumagai ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Growth Phase ◽

Exponential Growth ◽

Minimal Medium ◽

Exponential Growth Phase ◽

E Coli ◽

Genes Encoding ◽

K 12 ◽

Utilization Pathway

ABSTRACT The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP + strain absorbed up to 5 mM putrescine from the medium, but a ΔpuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

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Pyruvate Formate Lyase and Acetate Kinase Are Essential for Anaerobic Growth of Escherichia coli on Xylose

Journal of Bacteriology ◽

10.1128/jb.186.22.7593-7600.2004 ◽

2004 ◽

Vol 186 (22) ◽

pp. 7593-7600 ◽

Cited By ~ 96

Author(s):

Adnan Hasona ◽

Youngnyun Kim ◽

F. G. Healy ◽

L. O. Ingram ◽

K. T. Shanmugam

Keyword(s):

Escherichia Coli ◽

Minimal Medium ◽

High Capacity ◽

Redox Balance ◽

Anaerobic Growth ◽

Acetate Kinase ◽

Anaerobic Conditions ◽

Pyruvate Formate Lyase ◽

E Coli ◽

Xylose Transport

ABSTRACT During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

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Effects of Sodium Octanoate, Acylated Ghrelin, and Desacylated Ghrelin on the Growth of Genetically Engineered Escherichia Coli

Journal of Medical Biochemistry ◽

10.2478/v10011-011-0020-8 ◽

2011 ◽

Vol 30 (4) ◽

pp. 328-333 ◽

Cited By ~ 3

Author(s):

Suleyman Aydin ◽

Sebnem Erenler ◽

Yalcin Kendir

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Caprylic Acid ◽

Minimal Medium ◽

Genetically Engineered ◽

Acylated Ghrelin ◽

Sodium Octanoate ◽

Cell Counts ◽

The Media ◽

Desacylated Ghrelin

Effects of Sodium Octanoate, Acylated Ghrelin, and Desacylated Ghrelin on the Growth of Genetically EngineeredEscherichia ColiAcylated ghrelin is a 28-amino acid peptide hormone bearing a fatty acid group based on octanoic acid (caprylic acid) at the serine which is located at position 3 and at the N-terminus. If this fatty acid is cleaved from acylated ghrelin, the remaining peptide is referred to as desacylated ghrelin. Free fatty acids (FFAs) can kill or inhibit the growth of bacteria. The purpose of this study was to test this ability using acylated ghrelin, desacylated ghrelin, and sodium octanoate (caprylic acid) as carbon sources for the genetically engineeredEscherichia colistrains MK79 and MK57. For this experimental work, minimal medium was modified by replacing glucose with equal concentrations of acylated ghrelin, desacylated ghrelin, or sodium octanoate. Bacterial optical density, viability, alpha-amylase production, plasmid stability and pH of the growth medium were measured during these experiments. The media that allowed most growth, based on viable cell counts and the OD600of MK79, was minimal medium, followed by the medium containing desacylated ghrelin or acylated ghrelin, and finally the medium containing sodium octanoate. The same order was observed for MK57. Neither of the strains lost plasmids during the entire course of each experiment. There was also little change in the pH of any of the media used for both strains. These results suggest that sodium octanoate, acylated ghrelin, and desacylated ghrelin, when compared with minimal medium, inhibitEscherichia coligrowth. Proliferation was lowest when sodium octanoate was used as the carbon source, followed by acylated ghrelin and desacylated ghrelin. Therefore, the acylated ghrelin found previously in human saliva might help to inhibit pathogenic microorganisms, and acylated ghrelin levels below a critical concentration in saliva could result in an increased risk of oral infection.

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Medium Plays a Role in Determining Expression of acrB, marA, and soxS in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.1071-1074.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 1071-1074 ◽

Cited By ~ 14

Author(s):

Andrew M. Bailey ◽

Mark A. Webber ◽

Laura J. V. Piddock

Keyword(s):

Escherichia Coli ◽

Minimal Medium ◽

Rich Medium ◽

Growth Phases ◽

Minimal Media ◽

Logarithmic Growth

ABSTRACT Analysis of expression of acrB, marA, and soxS in rich and minimal media, at early and late logarithmic growth phases, showed that acrB had increased expression in minimal medium compared to rich medium, but expression decreased dose dependently upon exposure to ciprofloxacin.

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Inactivation of chloramphenicol by Gram-negative microorganisms

Canadian Journal of Microbiology ◽

10.1139/m68-150 ◽

1968 ◽

Vol 14 (8) ◽

pp. 891-899 ◽

Cited By ~ 9

Author(s):

David Sompolinsky ◽

Ruth Ziegler-Schlomowitz ◽

Dora Herczog

Keyword(s):

Escherichia Coli ◽

Growth Medium ◽

Minimal Medium ◽

Resistance Factor ◽

The Other ◽

Gram Negative ◽

Resistant Strains ◽

Gram Negative Organisms ◽

High Level ◽

Level Resistance

Two derivative strains of Escherichia coli with high-level resistance to chloramphenicol, one carrying an episomal resistance factor and the other a chromosomal mutant, were both shown to be potent inactivators of the drug. When 1 mM chloramphenicol was added to an exponential culture in minimal medium, growth was halted until 85–90% of the drug was inactivated by acylation. At this state the drug was essentially monoacylated. During and after growth, esterification of the second alcoholic group occurred, though at a slower rate. Arylamines, in amounts up to 10% of chloramphenicol equivalents, were demonstrated in the growth medium after 1–3 days' incubation.With an acetateless mutant of Escherichia coli K12, carrying a resistance factor, it was shown that 5–6 moles of acetate was consumed for every mole of chloramphenicol acylated.Inactivation of chloramphenicol by Gram-negative organisms from infections in hospitalized patients was also examined. Among 103 strains susceptible to chloramphenicol, none produced considerable amounts of chloramphenicol esters. The same was the case with 14 resistant strains of Pseudomonas. Of 134 other resistant organisms examined, including strains of Escherichia, Proteus, Klebsiella, Salmonella, and Shigella, 133 were producers of chloramphenicol esters, and in most cases the drug was partly or entirely diacylated.

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The ssu Locus Plays a Key Role in Organosulfur Metabolism in Pseudomonas putidaS-313

Journal of Bacteriology ◽

10.1128/jb.182.10.2869-2878.2000 ◽

2000 ◽

Vol 182 (10) ◽

pp. 2869-2878 ◽

Cited By ~ 55

Author(s):

Antje Kahnert ◽

Paul Vermeij ◽

Claudia Wietek ◽

Peter James ◽

Thomas Leisinger ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Related Species ◽

Minimal Medium ◽

Flavin Mononucleotide ◽

Flavin Reductase ◽

Sulfate Ester ◽

Two Component ◽

Aromatic Sulfonates ◽

Transposon Insertions

ABSTRACT Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded byssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.

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