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Engineering CHO cell metabolism for growth in galactose.

Jiménez NE, Wilkens CA, Gerdtzen ZP - BMC Proc (2011)

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, 8370448, Chile ; Millennium Institute for Cell Dynamics and Biotechnology: a Centre for Systems Biology, University of Chile, Santiago, 8370448, Chile.

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In this work we aim at identifying culture conditions that extend the culture's viability for tPA producing CHO cells in media with combined glucose and galactose as carbon sources... When glucose is depleted GG6/14 culture enters a second metabolic stage where no significant growth is observed and galactose is consumed along with extracellular lactate and alanine... This is consistent with lactate and alanine being used as a supplementary pyruvate source to support energy metabolism associated with cellular maintenance... Metabolic flux analysis (MFA) was performed, considering the main reactions of the central glucose and galactose metabolism... Figure 1(a) shows results for two time-points for each culture, an early (50 h) and a late time-point (100 h)... In the pyruvate node carbon molecules are channeled either towards the TCA cycle, or in the direction of secondary metabolite synthesis such as lactate and alanine... Fluxes from the central carbon metabolism are reduced several times in the late time point for both cultures, except for the Pyr-AcCoA reaction, which plays a central role in the regulation of mammalian metabolism by connecting glycolysis with the TCA cycle... Since galactose consumption appears to be limiting cell growth in GG6/14, there is an open possibility to improve cell growth during galactose consumption while maintaining low lactate production by over-expressing genes involved in galactose metabolism... Before transfection, mutation of a dileucine motif to alanine is required to allow the expression of this transporter in the plasmatic membrane... For that purpose an strategy using PCR with primers that include this substitutions is currently undergoing... When CHO cells are cultured with a combination of glucose and galactose it is possible to achieve extended viability along with a metabolic shift towards lactate consumption, which is triggered when the second hexose is consumed... Metabolism can be modified through cell engineering to increase cell growth while maintaining low lactate production rates, enabling cells to utilize alternative carbon sources.

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Experimental results and metabolic flux distribution. (a) Metabolic Flux distribution between G20 (red) and GG6/14 (blue) at 50 h and 100 h in culture. Cell density, glucose and lactate concentration in GG6/14  (b) and Gal20 media (c). (▪) CHO tPA,(●) CHO tPA-pcDNA3.1(+), (♦) CHO tPA-GALK1, glucose (▪,♦), lactate (▲,●).
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Figure 1: Experimental results and metabolic flux distribution. (a) Metabolic Flux distribution between G20 (red) and GG6/14 (blue) at 50 h and 100 h in culture. Cell density, glucose and lactate concentration in GG6/14 (b) and Gal20 media (c). (▪) CHO tPA,(●) CHO tPA-pcDNA3.1(+), (♦) CHO tPA-GALK1, glucose (▪,♦), lactate (▲,●).

Mentions: Metabolic flux analysis (MFA) was performed, considering the main reactions of the central glucose and galactose metabolism. Figure 1(a) shows results for two time-points for each culture, an early (50 h) and a late time-point (100 h). For GG6/14, the columns represent glucose and galactose consumption stages, respectively. In the pyruvate node carbon molecules are channeled either towards the TCA cycle, or in the direction of secondary metabolite synthesis such as lactate and alanine. Fluxes from the central carbon metabolism are reduced several times in the late time point for both cultures, except for the Pyr-AcCoA reaction, which plays a central role in the regulation of mammalian metabolism by connecting glycolysis with the TCA cycle. In late culture stages where hexose uptake is low, cell metabolism is directed towards maintaining TCA cycle fluxes in order to obtain energy. To achieve this in GG6/14, galactose and lactate are used as an additional carbon source.


Engineering CHO cell metabolism for growth in galactose.

Jiménez NE, Wilkens CA, Gerdtzen ZP - BMC Proc (2011)

Experimental results and metabolic flux distribution. (a) Metabolic Flux distribution between G20 (red) and GG6/14 (blue) at 50 h and 100 h in culture. Cell density, glucose and lactate concentration in GG6/14  (b) and Gal20 media (c). (▪) CHO tPA,(●) CHO tPA-pcDNA3.1(+), (♦) CHO tPA-GALK1, glucose (▪,♦), lactate (▲,●).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285024&req=5

Figure 1: Experimental results and metabolic flux distribution. (a) Metabolic Flux distribution between G20 (red) and GG6/14 (blue) at 50 h and 100 h in culture. Cell density, glucose and lactate concentration in GG6/14 (b) and Gal20 media (c). (▪) CHO tPA,(●) CHO tPA-pcDNA3.1(+), (♦) CHO tPA-GALK1, glucose (▪,♦), lactate (▲,●).
Mentions: Metabolic flux analysis (MFA) was performed, considering the main reactions of the central glucose and galactose metabolism. Figure 1(a) shows results for two time-points for each culture, an early (50 h) and a late time-point (100 h). For GG6/14, the columns represent glucose and galactose consumption stages, respectively. In the pyruvate node carbon molecules are channeled either towards the TCA cycle, or in the direction of secondary metabolite synthesis such as lactate and alanine. Fluxes from the central carbon metabolism are reduced several times in the late time point for both cultures, except for the Pyr-AcCoA reaction, which plays a central role in the regulation of mammalian metabolism by connecting glycolysis with the TCA cycle. In late culture stages where hexose uptake is low, cell metabolism is directed towards maintaining TCA cycle fluxes in order to obtain energy. To achieve this in GG6/14, galactose and lactate are used as an additional carbon source.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, 8370448, Chile ; Millennium Institute for Cell Dynamics and Biotechnology: a Centre for Systems Biology, University of Chile, Santiago, 8370448, Chile.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

In this work we aim at identifying culture conditions that extend the culture's viability for tPA producing CHO cells in media with combined glucose and galactose as carbon sources... When glucose is depleted GG6/14 culture enters a second metabolic stage where no significant growth is observed and galactose is consumed along with extracellular lactate and alanine... This is consistent with lactate and alanine being used as a supplementary pyruvate source to support energy metabolism associated with cellular maintenance... Metabolic flux analysis (MFA) was performed, considering the main reactions of the central glucose and galactose metabolism... Figure 1(a) shows results for two time-points for each culture, an early (50 h) and a late time-point (100 h)... In the pyruvate node carbon molecules are channeled either towards the TCA cycle, or in the direction of secondary metabolite synthesis such as lactate and alanine... Fluxes from the central carbon metabolism are reduced several times in the late time point for both cultures, except for the Pyr-AcCoA reaction, which plays a central role in the regulation of mammalian metabolism by connecting glycolysis with the TCA cycle... Since galactose consumption appears to be limiting cell growth in GG6/14, there is an open possibility to improve cell growth during galactose consumption while maintaining low lactate production by over-expressing genes involved in galactose metabolism... Before transfection, mutation of a dileucine motif to alanine is required to allow the expression of this transporter in the plasmatic membrane... For that purpose an strategy using PCR with primers that include this substitutions is currently undergoing... When CHO cells are cultured with a combination of glucose and galactose it is possible to achieve extended viability along with a metabolic shift towards lactate consumption, which is triggered when the second hexose is consumed... Metabolism can be modified through cell engineering to increase cell growth while maintaining low lactate production rates, enabling cells to utilize alternative carbon sources.

No MeSH data available.