BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects

Sara Pereira, Daniel Ley, Mikkel Schubert, Lise Marie Grav, Helene Faustrup Kildegaard, Mikael Rørdam Andersen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality. BCAA catabolism also hampers the usage of BCAAs for protein synthesis. In this work we studied the effects of disrupting the genes responsible for the first step of BCAA catabolism: branched chain aminotransferase 1 (Bcat1) and branched chain aminotransferase 2 (Bcat2). We evaluated the effect of disrupting the genes individually and in combination, and examined the effects in producer and non-producer host cells. Our experiments show that Bcat1 disruption improves cell growth in producer cells, but not in non-producers. Conversely, Bcat2 has a minor negative effect on growth in producer cells, and none in non-producers. Combined Bcat1 and Bcat2 disruption improves growth in producer cells. By-product metabolism is cell line-, clone- and producer-dependent. Overall, our results show that the effects of targeting Bcat1 and/or Bcat2 are cell line-dependent, and seemingly linked to the burden of recombinant protein expression.
Original languageEnglish
JournalJournal of Biotechnology
Volume306
Pages (from-to)24-31
Number of pages8
ISSN0168-1656
DOIs
Publication statusPublished - 2019

Keywords

  • Chinese hamster ovary cells
  • Branched-chain amino acids
  • Nutrient metabolism
  • By-product
  • CRISPR/Cas9

Cite this

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title = "BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects",
abstract = "In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality. BCAA catabolism also hampers the usage of BCAAs for protein synthesis. In this work we studied the effects of disrupting the genes responsible for the first step of BCAA catabolism: branched chain aminotransferase 1 (Bcat1) and branched chain aminotransferase 2 (Bcat2). We evaluated the effect of disrupting the genes individually and in combination, and examined the effects in producer and non-producer host cells. Our experiments show that Bcat1 disruption improves cell growth in producer cells, but not in non-producers. Conversely, Bcat2 has a minor negative effect on growth in producer cells, and none in non-producers. Combined Bcat1 and Bcat2 disruption improves growth in producer cells. By-product metabolism is cell line-, clone- and producer-dependent. Overall, our results show that the effects of targeting Bcat1 and/or Bcat2 are cell line-dependent, and seemingly linked to the burden of recombinant protein expression.",
keywords = "Chinese hamster ovary cells, Branched-chain amino acids, Nutrient metabolism, By-product, CRISPR/Cas9",
author = "Sara Pereira and Daniel Ley and Mikkel Schubert and Grav, {Lise Marie} and Kildegaard, {Helene Faustrup} and Andersen, {Mikael R{\o}rdam}",
year = "2019",
doi = "10.1016/j.jbiotec.2019.08.017",
language = "English",
volume = "306",
pages = "24--31",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier",

}

BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects. / Pereira, Sara; Ley, Daniel; Schubert, Mikkel; Grav, Lise Marie; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam.

In: Journal of Biotechnology, Vol. 306, 2019, p. 24-31.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects

AU - Pereira, Sara

AU - Ley, Daniel

AU - Schubert, Mikkel

AU - Grav, Lise Marie

AU - Kildegaard, Helene Faustrup

AU - Andersen, Mikael Rørdam

PY - 2019

Y1 - 2019

N2 - In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality. BCAA catabolism also hampers the usage of BCAAs for protein synthesis. In this work we studied the effects of disrupting the genes responsible for the first step of BCAA catabolism: branched chain aminotransferase 1 (Bcat1) and branched chain aminotransferase 2 (Bcat2). We evaluated the effect of disrupting the genes individually and in combination, and examined the effects in producer and non-producer host cells. Our experiments show that Bcat1 disruption improves cell growth in producer cells, but not in non-producers. Conversely, Bcat2 has a minor negative effect on growth in producer cells, and none in non-producers. Combined Bcat1 and Bcat2 disruption improves growth in producer cells. By-product metabolism is cell line-, clone- and producer-dependent. Overall, our results show that the effects of targeting Bcat1 and/or Bcat2 are cell line-dependent, and seemingly linked to the burden of recombinant protein expression.

AB - In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality. BCAA catabolism also hampers the usage of BCAAs for protein synthesis. In this work we studied the effects of disrupting the genes responsible for the first step of BCAA catabolism: branched chain aminotransferase 1 (Bcat1) and branched chain aminotransferase 2 (Bcat2). We evaluated the effect of disrupting the genes individually and in combination, and examined the effects in producer and non-producer host cells. Our experiments show that Bcat1 disruption improves cell growth in producer cells, but not in non-producers. Conversely, Bcat2 has a minor negative effect on growth in producer cells, and none in non-producers. Combined Bcat1 and Bcat2 disruption improves growth in producer cells. By-product metabolism is cell line-, clone- and producer-dependent. Overall, our results show that the effects of targeting Bcat1 and/or Bcat2 are cell line-dependent, and seemingly linked to the burden of recombinant protein expression.

KW - Chinese hamster ovary cells

KW - Branched-chain amino acids

KW - Nutrient metabolism

KW - By-product

KW - CRISPR/Cas9

U2 - 10.1016/j.jbiotec.2019.08.017

DO - 10.1016/j.jbiotec.2019.08.017

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