Gene switch for l‐glucose‐induced biopharmaceutical production in mammalian cells. Issue 6 (18th March 2021)

• Record Type: Journal Article
• Authors: Strittmatter, Tobias; Egli, Sabina; Bertschi, Adrian; Plieninger, Richard; Bojar, Daniel; Xie, Mingqi; Fussenegger, Martin
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 6(2021)
• Page Start: 2220
• Page End: 2233
• Publication Date: 2021-03-18
• Subjects: d‐idonate -- LgdA -- l‐glucose -- LgnH -- LgnI -- LgnR -- SMIT1 -- synthetic biology; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27730
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: 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. Abstract : l ‐Glucose is an artificial sweetener which is inert to human metabolism making it suitable for orthogonal induction of gene expression. To engineer l ‐glucose‐controlled gene switches, Strittmatter and coworkers expressed enzymes from Paraccocus species 43P in mammalian cells to convert l ‐glucose into d ‐idonate which binds to bacterial regulator protein LgnR causing it to dissociate from its respective operator sequence (OLgnR ). By fusing silencing (KRAB) or activating domains (VP16) to LgnR, ON‐ and OFF‐switches were designed to enable l ‐glucose‐induced expression of biopharmaceutical rituximab.