De novo design of programmable inducible promoters

Journal Article · 25 September 2019 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkz772 · OSTI ID:1566242

Liu, Xiangyang ^[1]; Gupta, Sanjan T. P. ^[2]; Bhimsaria, Devesh ^[3]; Reed, Jennifer L. ^[2]; Rodríguez-Martínez, José A.  ^[4]; Ansari, Aseem Z. ^[5]; Raman, Srivatsan  ^[6]

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1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA
2. The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
3. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
4. Department of Biology, University of Puerto Rico-Rio Piedras, San Juan, PR 00925, USA
5. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA
6. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA, The Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA, Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

• Sponsoring Organization: USDOE
• Grant/Contract Number: SC0018409; FC02-07ER64494
• OSTI ID: 1566242
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 19 Vol. 47; ISSN 0305-1048
• Publisher: Oxford University PressCopyright Statement
• Country of Publication: United Kingdom
• Language: English