Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA

Rhee, Jong Il; Bode, Jens; Ricci, Juan C. Díaz; Poock, Daniel; Weigel, B.; Kretzmer, G.; Schügerl, K.

doi:10.1016/s0168-1656(97)00058-8

Cited by 24 publications

(13 citation statements)

References 27 publications

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“…This kinetic effect predicted by the model may be used to produce this harmful protein under conditions that guarantees the viability of the host. However, since a < 20 min is a doubling time unattainable by E. coli with a simple homogeneous reactor, we may foresee that if a different type of reactor is used, such as an immobilized cell reactor or a two-stage cascade reactor, such as that reported by Rhee et al (1997) but with biomass feedback, the cultural conditions would be met so that the process would then be feasible.…”

Section: Discussionmentioning

confidence: 99%

Modeling of the controlled expression of a harmful protein by a three‐plasmid harboring system

Unzaga

Ricci

Rhee

et al. 2002

Biotech & Bioengineering

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A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.

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Section: Discussionmentioning

confidence: 99%

Modeling of the controlled expression of a harmful protein by a three‐plasmid harboring system

Unzaga

Ricci

Rhee

et al. 2002

Biotech & Bioengineering

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“…The three-plasmid system was cultivated in shaked¯asks and stirred tank reactors in various media in batch and continuous operation [35] and in a continuously operated two-stage cascade with cell growth in the ®rst stage and with induction of the gene expression in the second stage [36].…”

Section: Description Of the Biological Systemmentioning

confidence: 99%

“…On account of the missing experimental data for a large scale computer model in the genetic level, the presented simulations were performed by a four-compartment model which is based on the measured data [35,36]. The aim of the mathematical simulation was to describe the variations of the concentrations of glucose, acetate, cell mass, the intracellular proteins and the three plasmids during the cultivation.…”

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Mathematical simulation of the growth of a three plasmid harboring Escherichia coli JM109 strain and the production of the fusion protein EcoRI::SPA with a four-compartment model

Rhee

Schügerl

1998

Bioprocess Engineering

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In the previous papers [35,36] the process variables of plasmid-free, one-, two-and three-plasmid harboring E. coli JM109 cells were investigated in batch and continuous cultivation as a function of the medium composition, plasmid content, dilution rate and cultivation (generation) time. In the present paper the growth of the recombinant E. coli JM109 [pEcoR4, pRK248cI, pMTC48] and the production of the fusion protein EcoRI::SPA are simulated by using a four-compartment model, consisting of the active cell components (ribosomes, mRNA, tRNA, and others) (A), the structure forming materials and chromosomal DNA (Z), the plasmid-DNA (G) and the recombinant enzyme protein (E). At the ®rst time, all of the three plasmids: the production plasmid (Gp), the repressor plasmid (Gr) and the protection plasmid (Gs) are taken into account in the plasmid DNA-compartment of the model. The calculated and measured courses of the cell mass, the concentrations of glucose and acetate, and the products as well as the particular plasmids agree well. List of symbolsA active cell compartment [g (g X) A1 ] Ac acetate concentration [g l A1 ] E enzyme protein compartment; Ep for fusion protein, Er for repressor protein, Es for methylase [g (g X) A1 ] F¯ow rate [l h A1 ] G plasmid-DNA compartment; Gp for production plasmid, Gr for repressor plasmid, Gs for protection plasmidextracellular substrate concentration [g l A1 ] S Ã intracellular substrate concentration [g l A1 ] t cultivation time [h] t f the time ®lled up the second reactor with medium [h] V reactor volume [l] X cell mass [g l A1 ] X i intracellular concentration of the i-th component [g (g X) A1 ] y A,Z stoichiometric coef®cients for the formation of components Z compartment for structure forming material [g (g X) A1 ] l speci®c growth rate [h A1 ] g stoichiometric coef®cients for acetate formation x segregation coef®cient of the production plasmid 1 index for the ®rst reactor 2 index for the second reactor 20 index for the¯ow of induction medium to the second reactor + exponent for the three plasmids harbouring cells A exponent for the two plasmids harbouring cells 1 Introduction Several researcher developed mathematical models for the description of the growth and host plasmid interaction in recombinant E. coli [13, 38]. One can distinguish between small and large structured and population models [2]. In small structured models the cell mass and biological properties of the cells lumped into few compartments. A four-compartment model was presented by Nielsen et al. [24], which was later improved [26] based on the lactobacillus model [25]. An eight-compartment model was formulated by Bentley and Kompala [3]. Large scale computer models containing many variables and parameters represent an attempt to calculate, in systematic and coordinated fashion, the consequences of many simultaneous interactions within the cell, sometimes on the known molecular mechanism [2]. Such models were developed by Lee and Bailey [16±19, 21]. These models make it possible to simulate the cells growth, ...

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“…For bacterial and yeast hosts, loss of recombinant DNA may occur as a result of plasmid loss during cell division. Plasmid-bearing cells may have lower growth rates than plasmid-free cells because of the energy and resources required to maintain and express the extra DNA in the cell (Glick, 1995;Rhee et al, 1997) and thus any selection pressure which favors strains with growth rate advantages will favor plasmid-free cells. In fungal cells, recombinant DNA is integrated into the genome.…”

Section: Introductionmentioning

confidence: 99%

Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in chemostat culture

Wiebe

Robson

Shuster

et al. 2001

Biotech & Bioengineering

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Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates.

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Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA

Cited by 24 publications

References 27 publications

Modeling of the controlled expression of a harmful protein by a three‐plasmid harboring system

Modeling of the controlled expression of a harmful protein by a three‐plasmid harboring system

Mathematical simulation of the growth of a three plasmid harboring Escherichia coli JM109 strain and the production of the fusion protein EcoRI::SPA with a four-compartment model

Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in chemostat culture

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