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Gene switch for l ‐glucose‐induced biopharmaceutical production in mammalian cells
Author(s) -
Strittmatter Tobias,
Egli Sabina,
Bertschi Adrian,
Plieninger Richard,
Bojar Daniel,
Xie Mingqi,
Fussenegger Martin
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27730
Subject(s) - hek 293 cells , transfection , chinese hamster ovary cell , activator (genetics) , microbiology and biotechnology , chemistry , transcription factor , transgene , biology , cell culture , biochemistry , gene , receptor , genetics
In this study, we designed and built a gene switch that employs metabolically inert l ‐glucose to regulate transgene expression in mammalian cells via d ‐idonate‐mediated control of the bacterial regulator LgnR. To this end, we engineered a metabolic cascade in mammalian cells to produce the inducer molecule d ‐idonate from its precursor l ‐glucose by ectopically expressing the Paracoccus species 43P‐derived catabolic enzymes LgdA, LgnH , and LgnI . To obtain ON‐ and OFF‐switches, we fused LgnR to the human transcriptional silencer domain Krüppel associated box (KRAB) and the viral trans‐activator domain VP16, respectively. Thus, these artificial transcription factors KRAB‐LgnR or VP16‐LgnR modulated cognate promoters containing LgnR‐specific binding sites in a d ‐idonate‐dependent manner as a direct result of l ‐glucose metabolism. In a proof‐of‐concept experiment, we show that the switches can control production of the model biopharmaceutical rituximab in both transiently and stably transfected HEK‐293T cells, as well as CHO‐K1 cells. Rituximab production reached 5.9 µg/ml in stably transfected HEK‐293T cells and 3.3 µg/ml in stably transfected CHO‐K1 cells.