Transcriptional regulation is of great significance for cells to maintain homeostasis and, meanwhile, represents an innovative but less explored means to control biological processes in synthetic biology and bioengineering. Herein we devised a T7 RNA polymerase (T7RNAP) variant through replacing an essential lysine located in the catalytic core (K631) with N-acetyl-l-lysine (AcK) via genetic code expansion. This T7RNAP variant requires the deacetylase activity of NAD-dependent sirtuins to recover its enzymatic activities and thereby sustains sirtuin-dependent transcription of the gene of interest in live cells including bacteria and mammalian cells as well as in in vitro systems. This T7RNAP variant could link gene transcription to sirtuin expression and NAD availability, thus holding promise to support some relevant research.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1021/acssynbio.3c00607 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A Sirtuin-Dependent T7 RNA Polymerase Variant.
ACS Synth Biol
January 2024
Frontiers Science Center for Synthetic Biology, Tianjin Key Laboratory of Function and Application of Biological Macromolecular Structures, School of Life Sciences, Tianjin University, Tianjin 300072, China.
Transcriptional regulation is of great significance for cells to maintain homeostasis and, meanwhile, represents an innovative but less explored means to control biological processes in synthetic biology and bioengineering. Herein we devised a T7 RNA polymerase (T7RNAP) variant through replacing an essential lysine located in the catalytic core (K631) with N-acetyl-l-lysine (AcK) via genetic code expansion. This T7RNAP variant requires the deacetylase activity of NAD-dependent sirtuins to recover its enzymatic activities and thereby sustains sirtuin-dependent transcription of the gene of interest in live cells including bacteria and mammalian cells as well as in in vitro systems.
View Article and Find Full Text PDFTunable T7 Promoter Orthogonality on T7RNAP for -Aconitate Decarboxylase Evolution via Base Editor and Screening from Itaconic Acid Biosensor.
ACS Synth Biol
October 2023
Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan.
The deaminase-fused T7 RNA polymerase (T7RNAP) presents a promising toolkit for target-specific enzyme evolution, offering the unique advantage of simultaneous DNA modification and screening. Previous studies have reported the mutation efficiency of base editors relying on different resources. In contrast, the mechanism underlying the T7RNAP/T7 system is well-understood.
View Article and Find Full Text PDFTargeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter evolution.
Appl Environ Microbiol
October 2023
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e).
View Article and Find Full Text PDFRNA-mediated Directed Evolution in Yeast.
Bio Protoc
March 2022
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.
Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to search variant landscapes based on or mutagenesis, to identify and select new-to-nature and optimized properties in biomolecules.
View Article and Find Full Text PDFDynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.
ACS Synth Biol
November 2017
Department of Biosystems Science and Engineering (D-BSSE), ETH-Zürich , Mattenstrasse 26, 4058 Basel, Switzerland.
Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!