Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli

Calleja, Daniel; Fernández-Castañé, Alfred; Pasini, Martina; Mas, Carles de; López-Santı́n, Josep

doi:10.1016/j.bej.2014.08.017

Cited by 3 publications

(4 citation statements)

References 32 publications

(60 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The IPTG uptake model (Calleja et al, ) is employed here with some improvements, which slightly modify the values of the parameters. It requires as inputs the biomass and IPTG concentration at induction time and uses the volume and biomass concentration predicted by the growth equations.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Simulation and prediction of protein production in fed‐batch E. coli cultures: An engineering approach

Calleja

Kavanagh

Mas

et al. 2015

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

An overall model describing the dynamic behavior of fed-batch E. coli processes for protein production has been built, calibrated and validated. Using a macroscopic approach, the model consists of three interconnected blocks allowing simulation of biomass, inducer and protein concentration profiles with time. The model incorporates calculation of the extra and intracellular inducer concentration, as well as repressor-inducer dynamics leading to a successful prediction of the product concentration. The parameters of the model were estimated using experimental data of a rhamnulose-1-phosphate aldolase-producer strain, grown under a wide range of experimental conditions. After validation, the model has successfully predicted the behavior of different strains producing two different proteins: fructose-6-phosphate aldolase and ω-transaminase. In summary, the presented approach represents a powerful tool for E. coli production process simulation and control.

show abstract

Section: Resultsmentioning

confidence: 99%

“…With regards to inducer transport, our research group has recently developed a model to describe and simulate intracellular IPTG concentrations with time (Calleja et al, ), this model was calibrated with previously published experimental IPTG measurements (Fernández et al, ).…”

Section: Introductionmentioning

confidence: 99%

Simulation and prediction of protein production in fed‐batch E. coli cultures: An engineering approach

Calleja

Kavanagh

Mas

et al. 2015

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

show abstract

“…For experiments performed in the Biostat® B bioreactor, a volume of 80 mL of the inoculum culture was added to 720 mL of DM in a 2-L vessel (Vidal et al, 2005 ; Calleja et al, 2014 ). pO 2 set point was fixed at 60% saturation, and pO 2 was controlled by adapting the stirring speed between 350 and 1120 rpm and by supplying air (enriched with pure oxygen when necessary) at a flow rate of 1.5 vvm.…”

Section: Methodsmentioning

confidence: 99%

“…The E. coli lac promoter is arguably the most extensively studied (Germán et al, 2019 ). Within the realm of E. coli expression, the T5 promoter system (and its derivatives) available from QIAGEN is one of the most popular choices for protein production (Fernández-Castané et al, 2012a ; Calleja et al, 2014 ). This IPTG-induced promoter has a high transcription rate that can only be tightly regulated in the presence of high levels of the Lac repressor protein (LacI), which is encoded by the lacI gene (Xu & Matthews, 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

Pasini

Fernández-Castañé

Caminal

et al. 2022

Journal of Industrial Microbiology and Biotechnology

Self Cite

View full text Add to dashboard Cite

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, capacity to efficiently synthesize the desired product and the use of selection markers that are acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of engineered strains—with different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA)—in high cell density cultures to evaluate their suitability to be used in bioprocess conditions that resemble industrial production. Results revealed that the first generation of engineered strain showed a 50 % reduction in fuculose-1-phosphate aldolase (FucA) production compared to the reference system (165 ± 13 mg FucA·g–1 DCW and 806 ± 12 AU·g–1 DCW) which is commercially available from QIAGEN and uses an antibiotic selection marker. The over-transcription glyA was found to be a major factor responsible for the metabolic burden leading to the decrease in FucA yield. The second- and third-generation of E. coli strains presented an increase in FucA production, being 202 ± 18 mg–1 FucA·g–1 DCW and 1176 ± 19 AU·g–1 DCW, and 1322 ± 19 AU·g–1 DCW and 245 ± 13 mg–1 FucA·g–1 DCW, respectively. Both strains presented a fitness improvement after the tuning of glyA expression levels and the deletion of the ampicillin resistance gene (bla) from the plasmid were carried out. The third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and is capable to produce FucA at similar levels compared to the original commercial expression system. Hence, our expression system possesses advantageous features compared to the commercial one and proved to have the potential to become an attractive platform for the production of recombinant proteins in a wide range of industrial biotechnology applications.

show abstract

Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of Escherichia coli—a rate limiting step in the induction of recombinant protein expression

Simas,

Pessoa Junior,

Long

2023

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts.

show abstract

Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli

Cited by 3 publications

References 32 publications

Simulation and prediction of protein production in fed‐batch E. coli cultures: An engineering approach

Simulation and prediction of protein production in fed‐batch E. coli cultures: An engineering approach

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of Escherichia coli—a rate limiting step in the induction of recombinant protein expression

Contact Info

Product

Resources

About