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Title: A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ 70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5'-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced throughmore » these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. Furthermore, these systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.« less

Authors:
ORCiD logo [1]; ORCiD logo [2];  [1];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States); Univ. of Colorado, Boulder, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Energy Efficiency and Renewable Energy (EERE), NREL Laboratory Directed Research and Development (LDRD); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Hydrogen and Fuel Cell Technologies Program (EE-3F)
OSTI Identifier:
1416722
Report Number(s):
NREL/JA-2700-70604
Journal ID: ISSN 2161-5063
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; cyanobacteria; ethylene-forming enzyme; promoter; protein expression; RBS; synthetic biology

Citation Formats

Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, and Yu, Jianping. A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803. United States: N. p., 2017. Web. doi:10.1021/acssynbio.7b00297.
Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, & Yu, Jianping. A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803. United States. doi:10.1021/acssynbio.7b00297.
Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, and Yu, Jianping. Tue . "A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803". United States. doi:10.1021/acssynbio.7b00297.
@article{osti_1416722,
title = {A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803},
author = {Wang, Bo and Eckert, Carrie and Maness, Pin -Ching and Yu, Jianping},
abstractNote = {Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5'-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced through these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. Furthermore, these systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.},
doi = {10.1021/acssynbio.7b00297},
journal = {ACS Synthetic Biology},
number = 1,
volume = 7,
place = {United States},
year = {Tue Dec 12 00:00:00 EST 2017},
month = {Tue Dec 12 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
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