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 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 thesemore »
- Authors:
-
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- National Renewable Energy Lab. (NREL), Golden, CO (United States); Univ. of Colorado, Boulder, CO (United States)
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office; 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:
- 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. https://doi.org/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. https://doi.org/10.1021/acssynbio.7b00297. https://www.osti.gov/servlets/purl/1416722.
@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}
}
Web of Science
Works referenced in this record:
Positive Regulation of psbA Gene Expression by cis-Encoded Antisense RNAs in Synechocystis sp. PCC 6803
journal, August 2012
- Sakurai, Isamu; Stazic, Damir; Eisenhut, Marion
- Plant Physiology, Vol. 160, Issue 2
Metabolic engineering of cyanobacteria for the synthesis of commodity products
journal, June 2015
- Angermayr, S. Andreas; Gorchs Rovira, Aleix; Hellingwerf, Klaas J.
- Trends in Biotechnology, Vol. 33, Issue 6
Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency
journal, January 2013
- Seo, Sang Woo; Yang, Jae-Seong; Kim, Inhae
- Metabolic Engineering, Vol. 15
Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803
journal, July 2015
- Ng, Andrew H.; Berla, Bertram M.; Pakrasi, Himadri B.
- Applied and Environmental Microbiology, Vol. 81, Issue 19
Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120
journal, December 1993
- Elhai, Jeff
- FEMS Microbiology Letters, Vol. 114, Issue 2
RBSDesigner: software for designing synthetic ribosome binding sites that yields a desired level of protein expression
journal, August 2010
- Na, D.; Lee, D.
- Bioinformatics, Vol. 26, Issue 20
Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology
journal, March 2010
- Huang, Hsin-Ho; Camsund, Daniel; Lindblad, Peter
- Nucleic Acids Research, Vol. 38, Issue 8
Photosynthetic conversion of carbon dioxide to ethylene by the recombinant cyanobacterium, Synechococcus sp. PCC 7942, which harbors a gene for the ethylene-forming enzyme of Pseudomonas syringae
journal, January 1997
- Sakai, Miho; Ogawa, Takahira; Matsuoka, Masayoshi
- Journal of Fermentation and Bioengineering, Vol. 84, Issue 5, p. 434-443
Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria
journal, March 2014
- Zhou, Jie; Zhang, Haifeng; Meng, Hengkai
- Scientific Reports, Vol. 4, Issue 1
Combinatorial optimization of cyanobacterial 2,3-butanediol production
journal, March 2014
- Oliver, John W. K.; Machado, Iara M. P.; Yoneda, Hisanari
- Metabolic Engineering, Vol. 22
Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2
journal, March 2013
- Wang, Bo; Pugh, Shawn; Nielsen, David R.
- Metabolic Engineering, Vol. 16
The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene
journal, April 2015
- Xiong, Wei; Morgan, John A.; Ungerer, Justin
- Nature Plants, Vol. 1, Issue 5
Sustained photosynthetic conversion of CO2 to ethylene in recombinant cyanobacterium Synechocystis 6803
journal, January 2012
- Ungerer, Justin; Tao, Ling; Davis, Mark
- Energy & Environmental Science, Vol. 5, Issue 10, p. 8998-9006
Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae
journal, October 1992
- Fukuda, Hideo; Ogawa, Takahira; Tazaki, Masato
- Biochemical and Biophysical Research Communications, Vol. 188, Issue 2
Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803
journal, November 2016
- Englund, Elias; Liang, Feiyan; Lindberg, Pia
- Scientific Reports, Vol. 6, Issue 1
An internal antisense RNA regulates expression of the photosynthesis gene isiA
journal, April 2006
- Duhring, U.; Axmann, I. M.; Hess, W. R.
- Proceedings of the National Academy of Sciences, Vol. 103, Issue 18
Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production
journal, December 2015
- Armshaw, Patricia; Carey, Dawn; Sheahan, Con
- Biotechnology for Biofuels, Vol. 8, Issue 1
Ethylene-forming enzyme and bioethylene production
journal, January 2014
- Eckert, Carrie; Xu, Wu; Xiong, Wei
- Biotechnology for Biofuels, Vol. 7, Issue 1
The plasticity of cyanobacterial carbon metabolism
journal, December 2017
- Xiong, Wei; Cano, Melissa; Wang, Bo
- Current Opinion in Chemical Biology, Vol. 41
Engineering of genetic control tools in Synechocystis sp. PCC 6803 using rational design techniques
journal, December 2015
- Albers, Stevan C.; Gallegos, Victor A.; Peebles, Christie A. M.
- Journal of Biotechnology, Vol. 216
Genetic and genomic analysis of RNases in model cyanobacteria
journal, January 2015
- Cameron, Jeffrey C.; Gordon, Gina C.; Pfleger, Brian F.
- Photosynthesis Research, Vol. 126, Issue 1
Automated design of synthetic ribosome binding sites to control protein expression
journal, October 2009
- Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
- Nature Biotechnology, Vol. 27, Issue 10, p. 946-950
Cyanobacterial-based approaches to improving photosynthesis in plants
journal, October 2012
- Zarzycki, Jan; Axen, Seth D.; Kinney, James N.
- Journal of Experimental Botany, Vol. 64, Issue 3
Effects of global transcription factor NtcA on photosynthetic production of ethylene in recombinant Synechocystis sp. PCC 6803
journal, June 2017
- Mo, Huilin; Xie, Xiaoman; Zhu, Tao
- Biotechnology for Biofuels, Vol. 10, Issue 1
Life and the Evolution of Earth's Atmosphere
journal, May 2002
- Kasting, J. F.
- Science, Vol. 296, Issue 5570
Ethylene production with engineered Synechocystis sp PCC 6803 strains
journal, February 2017
- Veetil, Vinod Puthan; Angermayr, S. Andreas; Hellingwerf, Klaas J.
- Microbial Cell Factories, Vol. 16, Issue 1
Enhanced limonene production in cyanobacteria reveals photosynthesis limitations
journal, November 2016
- Wang, Xin; Liu, Wei; Xin, Changpeng
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 50
Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate
journal, April 2017
- Zhang, Zhihong; Smart, Tristan J.; Choi, Hwanho
- Proceedings of the National Academy of Sciences, Vol. 114, Issue 18
Application of synthetic biology in cyanobacteria and algae
journal, September 2012
- Wang, Bo; Wang, Jiangxin; Zhang, Weiwen
- Frontiers in Microbiology, Vol. 3, Article No. 344
Amplified expression of a transcriptional pattern formed during development of Anabaena
journal, February 1993
- Wolk, C. Peter; Elhai, Jeff; Kuritz, Tanya
- Molecular Microbiology, Vol. 7, Issue 3
An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803
journal, January 2011
- Mitschke, Jan; Georg, Jens; Scholz, Ingeborg
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 5
Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002
journal, September 2014
- Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E.
- ACS Synthetic Biology, Vol. 4, Issue 5
Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist
journal, August 2017
- Martinez, Salette; Fellner, Matthias; Herr, Caitlyn Q.
- Journal of the American Chemical Society, Vol. 139, Issue 34
Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803
journal, January 2014
- Angermayr, S.; van der Woude, Aniek D.; Correddu, Danilo
- Biotechnology for Biofuels, Vol. 7, Issue 1
Enhancer activity of light-responsive regulatory elements in the untranslated leader regions of cyanobacterial psbA genes.
journal, December 1993
- Li, R.; Golden, S. S.
- Proceedings of the National Academy of Sciences, Vol. 90, Issue 24
Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence: Cyanobacterial Carbon Metabolism
journal, March 2017
- Wan, Ni; DeLorenzo, Drew M.; He, Lian
- Biotechnology and Bioengineering, Vol. 114, Issue 7
Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity.
journal, March 1985
- Brosius, J.; Erfle, M.; Storella, J.
- Journal of Biological Chemistry, Vol. 260, Issue 6
Strategies for achieving high-level expression of genes in Escherichia coli
journal, September 1996
- Makrides, S. C.
- Microbiological Reviews, Vol. 60, Issue 3
Works referencing / citing this record:
Regulatory systems for gene expression control in cyanobacteria
journal, January 2020
- Till, Petra; Toepel, Jörg; Bühler, Bruno
- Applied Microbiology and Biotechnology, Vol. 104, Issue 5
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology
journal, September 2019
- Gale, Grant A. R.; Schiavon Osorio, Alejandra A.; Mills, Lauren A.
- Microorganisms, Vol. 7, Issue 10
Citrate synthase from Synechocystis is a distinct class of bacterial citrate synthase
journal, April 2019
- Ito, Shoki; Koyama, Naoto; Osanai, Takashi
- Scientific Reports, Vol. 9, Issue 1
Progress and challenges in engineering cyanobacteria as chassis for light‐driven biotechnology
journal, August 2019
- Hitchcock, Andrew; Hunter, C. Neil; Canniffe, Daniel P.
- Microbial Biotechnology, Vol. 13, Issue 2
Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803
journal, March 2018
- Liu, Deng; Pakrasi, Himadri B.
- Microbial Cell Factories, Vol. 17, Issue 1
Diverse light responses of cyanobacteria mediated by phytochrome superfamily photoreceptors
journal, November 2018
- Wiltbank, Lisa B.; Kehoe, David M.
- Nature Reviews Microbiology, Vol. 17, Issue 1
Heterologous expression of bacterial natural product biosynthetic pathways
collection, January 2019
- Huo, Liujie; Hug, Joachim Josef; Fu, Chengzhang
- Universität des Saarlandes
The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux inSynechocystissp. PCC 6803
journal, August 2019
- Bartasun, Paulina; Prandi, Nicole; Storch, Marko
- PeerJ, Vol. 7
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology
journal, September 2019
- Gale, Grant A. R.; Schiavon Osorio, Alejandra A.; Mills, Lauren A.
- Microorganisms, Vol. 7, Issue 10
Engineering cyanobacteria for production of terpenoids
journal, November 2018
- Lin, Po-Cheng; Pakrasi, Himadri B.
- Planta, Vol. 249, Issue 1
New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering
journal, February 2019
- Santos-Merino, María; Singh, Amit K.; Ducat, Daniel C.
- Frontiers in Bioengineering and Biotechnology, Vol. 7
Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production
journal, June 2020
- Sebesta, Jacob; Peebles, Christie AM.
- Metabolic Engineering Communications, Vol. 10
Unlocking the photobiological conversion of CO 2 to ( R )-3-hydroxybutyrate in cyanobacteria
journal, January 2018
- Wang, Bo; Xiong, Wei; Yu, Jianping
- Green Chemistry, Vol. 20, Issue 16
Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO2
journal, May 2020
- Zhang, Li; Chen, Lei; Diao, Jinjin
- Biotechnology for Biofuels, Vol. 13, Issue 1
Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803
journal, November 2018
- Afrin, Shajia; Khan, Md. Rezaul Islam; Zhang, Weiyi
- Frontiers in Microbiology, Vol. 9
Photosynthetic production of the nitrogen-rich compound guanidine
journal, January 2019
- Wang, Bo; Dong, Tao; Myrlie, Aldon
- Green Chemistry, Vol. 21, Issue 11
State-of-the-Art Genetic Modalities to Engineer Cyanobacteria for Sustainable Biosynthesis of Biofuel and Fine-Chemicals to Meet Bio–Economy Challenges
journal, June 2019
- Khan, Aqib; Bilal, Muhammad; Mehmood, Shahid
- Life, Vol. 9, Issue 3
Recent advances in synthetic biology of cyanobacteria
journal, May 2018
- Sengupta, Annesha; Pakrasi, Himadri B.; Wangikar, Pramod P.
- Applied Microbiology and Biotechnology, Vol. 102, Issue 13
Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria
journal, April 2019
- Xia, Peng‐Fei; Ling, Hua; Foo, Jee Loon
- Biotechnology Journal, Vol. 14, Issue 6
Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803
journal, November 2018
- Afrin, Shajia; Khan, Md. Rezaul Islam; Zhang, Weiyi
- Frontiers in Microbiology, Vol. 9
Heterologous expression of bacterial natural product biosynthetic pathways
journal, January 2019
- Huo, Liujie; Hug, Joachim J.; Fu, Chengzhang
- Natural Product Reports, Vol. 36, Issue 10