DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630

Journal Article · · ACS Synthetic Biology
 [1];  [1]
  1. Washington Univ., St. Louis, MO (United States)

Rhodococcus opacus PD630 (R. opacus) is a non-model, gram-positive bacterium which holds promise as a biological catalyst for the conversion of lignocellulosic biomass into value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. Yet, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Moreover, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to constructing AND and NAND genetic logic gates. Additionally, three IPTG-inducible promoters were created by inserting LacI binding sites into newly-characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Lastly, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enabling complex and tunable gene regulation in this promising non-model host for bioproduction.

Research Organization:
Washington Univ., St. Louis, MO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
SC0018324
OSTI ID:
1545618
Journal Information:
ACS Synthetic Biology, Vol. 8, Issue 8; ISSN 2161-5063
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 18 works
Citation information provided by
Web of Science

References (52)

Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications journal August 2016
Improving fatty acids production by engineering dynamic pathway regulation and metabolic control journal July 2014
Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways journal December 2015
Programmable genetic circuits for pathway engineering journal December 2015
Synthesizing Biomolecule-Based Boolean Logic Gates journal December 2012
Enabling complex genetic circuits to respond to extrinsic environmental signals: Genetic Circuits to Sense Environmental Signals journal March 2017
Oxygen-responsive genetic circuits constructed in Synechocystis sp. PCC 6803 : Oxygen-Responsive Gene Circuits for journal September 2015
Genetic circuit design automation journal March 2016
Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803 journal March 2019
Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630 journal June 2017
Non-model model organisms journal June 2017
Challenges and Advances for Genetic Engineering of Non-model Bacteria and Uses in Consolidated Bioprocessing journal October 2017
Rhodococcus opacus strain PD630 as a new source of high-value single-cell oil? Isolation and characterization of triacylglycerols and other storage lipids journal May 2000
Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630 journal June 1996
Triacylglycerol Production from Corn Stover Using a Xylose-Fermenting Rhodococcus opacus Strain for Lignocellulosic Biofuels journal January 2014
Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630 journal February 2016
Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus journal September 2018
Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors journal January 2015
Lipid Production from Dilute Alkali Corn Stover Lignin by Rhodococcus Strains journal January 2017
Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630 journal January 2018
Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630 journal April 2018
The Interrelationship between Promoter Strength, Gene Expression, and Growth Rate journal October 2014
Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains journal June 2013
Engineering of a Xylose Metabolic Pathway in Rhodococcus Strains journal May 2012
lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA journal January 2012
Minimal length of the lactose operator sequence for the specific recognition by the lactose repressor. journal March 1977
Regulation of ribosomal RNA promoters with a synthetic lac operator. journal November 1984
Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. journal January 1984
Anatomy of Escherichia coli σ 70 promoters journal December 2006
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes journal May 1986
A Highly Efficient Cell-Free Protein Synthesis System from Escherichia coli journal August 1996
Recombinant protein expression in Escherichia coli journal October 1999
Manufacturing of recombinant therapeutic proteins in microbial systems journal February 2006
Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry journal October 2014
Kinetic and Thermodynamic Basis of Promoter Strength:  Multiple Steps of Transcription Initiation by T7 RNA Polymerase Are Modulated by the Promoter Sequence journal March 2002
Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants journal March 2013
Multi-Input Regulation and Logic with T7 Promoters in Cells and Cell-Free Systems journal October 2013
Mechanisms for activating bacterial RNA polymerase journal September 2010
Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor journal May 1991
Genetic programs constructed from layered logic gates in single cells journal October 2012
Isolation and characterization of the Rhodococcus opacus thiostrepton-inducible genes tipAL and tipAS : application for recombinant protein expression in Rhodococcus journal August 2004
The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci: Regulation of triacylglycerol accumulation in rhodococci journal November 2016
NpdR, a Repressor Involved in 2,4,6-Trinitrophenol Degradation in Rhodococcus opacus HL PM-1 journal January 2004
Construction of a BioBrick™ compatible vector system for Rhodococcus journal March 2017
Validation of two ribosomal RNA removal methods for microbial metatranscriptomics journal September 2010
Core element characterization of Rhodococcus promoters and development of a promoter-RBS mini-pool with different activity levels for efficient gene expression journal September 2018
Synthetic promoter libraries for Corynebacterium glutamicum journal January 2014
Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements journal January 2018
Enzymatic assembly of DNA molecules up to several hundred kilobases journal April 2009
The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse journal February 2008
Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids) journal October 1999
Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13 C-metabolite fingerprinting journal September 2015

Cited By (3)

Genetically encoded biosensors for lignocellulose valorization journal October 2019
Biosensing in Smart Engineered Probiotics journal January 2020
Genetically encoded biosensors for lignocellulose valorization journal October 2019

Figures / Tables (6)