Title: Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites

Abstract

Abstract Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering.

Authors:

Wang, Tiebin ^[1]; Tague, Nathan ^[2]; Whelan, Stephen A. ^[3]; Dunlop, Mary J.  ^[4]

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+ Show Author Affiliations

1. Molecular Biology, Cell Biology & Biochemistry, Boston University, Boston, MA 02215, USA, Biological Design Center, Boston University, Boston, MA 02215, USA
2. Biological Design Center, Boston University, Boston, MA 02215, USA, Biomedical Engineering, Boston University, Boston, MA 02215, USA
3. Chemistry, Boston University, Boston, MA 02215, USA
4. Molecular Biology, Cell Biology & Biochemistry, Boston University, Boston, MA 02215, USA, Biological Design Center, Boston University, Boston, MA 02215, USA, Biomedical Engineering, Boston University, Boston, MA 02215, USA

Publication Date:

Thu Dec 24 00:00:00 EST 2020

Research Org.:

Boston Univ., MA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)

OSTI Identifier:

1745084

Alternate Identifier(s):

OSTI ID: 1853173

Grant/Contract Number:  

SC0019387; 1804096

Resource Type:

Published Article

Journal Name:

Nucleic Acids Research

Additional Journal Information:

Journal Name: Nucleic Acids Research Journal Volume: 49 Journal Issue: 2; Journal ID: ISSN 0305-1048

Publisher:

Oxford University Press

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES