1. Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli
- Author
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Chiara Guidi, Lien De Wannemaeker, Jasmine De Baets, Wouter Demeester, Jo Maertens, Brecht De Paepe, and Marjan De Mey
- Subjects
Science & Technology ,HIGH-LEVEL EXPRESSION ,ENVELOPE STRESS ,Biology and Life Sciences ,sRNA-based genetic circuitry ,Bioengineering ,LOCALIZATION ,Industrial biotechnology ,Applied Microbiology and Biotechnology ,TRANSDUCTION ,OVERFLOW METABOLISM ,HFQ ,PATHWAY ,Biotechnology & Applied Microbiology ,FLUORESCENT PROTEIN ,Membrane proteins ,ACID ,Escherichia coli ,Life Sciences & Biomedicine ,SYSTEM ,Biotechnology - Abstract
Background Membrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential—as therapeutic drug targets or in microbial cell factory optimization—many challenges remain for efficient and functional expression in a host such as Escherichia coli. Results A dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general. Conclusions A dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress.
- Published
- 2022