CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Gordon, Gina C.; Korosh, Travis C.; Cameron, Jeffrey C.; Markley, Andrew L.; Begemann, Matthew B.; Pfleger, Brian F.

doi:10.1016/j.ymben.2016.07.007

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Journal Article · Fri Jul 29 00:00:00 EDT 2016 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2016.07.007· OSTI ID:1424612

Gordon, Gina C. ^[1]; Korosh, Travis C. ^[2]; Cameron, Jeffrey C. ^[3]; Markley, Andrew L. ^[2]; Begemann, Matthew B. ^[2]; Pfleger, Brian F. ^[2]

University of Wisconsin, Madison, WI (United States); University of Wisconsin
University of Wisconsin, Madison, WI (United States)
University of Colorado, Boulder, CO (United States)

Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. In this paper, we have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO₂. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. Finally, this tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

View Accepted Manuscript (DOE)

Research Organization:: University of Wisconsin, Madison, WI (United States)

Sponsoring Organization:: National Inst. of Health (NIH); National Science Foundation (NSF); USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0010329

OSTI ID:: 1424612

Alternate ID(s):: OSTI ID: 1436836

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 38; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

References (58)

Insights into the industrial growth of cyanobacteria from a model of the carbon-concentrating mechanism Clark, Ryan L.; Cameron, Jeffrey C.; Root, Thatcher W. AIChE Journal, Vol. 60, Issue 4 https://doi.org/10.1002/aic.14310	journal	January 2014
Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002 : Construction of New Synthetic Biology Tools Zess, Erin K.; Begemann, Matthew B.; Pfleger, Brian F. Biotechnology and Bioengineering, Vol. 113, Issue 2 https://doi.org/10.1002/bit.25713	journal	September 2015
A single-point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor Richter, Nina; Zienert, Anke; Hummel, Werner Engineering in Life Sciences, Vol. 11, Issue 1 https://doi.org/10.1002/elsc.201000151	journal	January 2011
Phycobilisomes of the Cyanobacterium Synechococcus SP. PCC 7002: Structure, Function, Assembly, and Expression Bryant, Donald A.; Zhou, Jianhui; Gasparich, Gail E. Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria https://doi.org/10.1007/978-1-4757-0893-6_17	book	January 1990
Metabolic design for cyanobacterial chemical synthesis Oliver, John W. K.; Atsumi, Shota Photosynthesis Research, Vol. 120, Issue 3 https://doi.org/10.1007/s11120-014-9997-4	journal	April 2014
Site-directed mutagenesis by overlap extension using the polymerase chain reaction Ho, Steffan N.; Hunt, Henry D.; Horton, Robert M. Gene, Vol. 77, Issue 1, p. 51-59 https://doi.org/10.1016/0378-1119(89)90358-2	journal	April 1989
Regulation of ammonium assimilation in cyanobacteria Muro-Pastor, M. Isabel; Florencio, Francisco J. Plant Physiology and Biochemistry, Vol. 41, Issue 6-7 https://doi.org/10.1016/S0981-9428(03)00066-4	journal	June 2003
Glycogen synthesis is a required component of the nitrogen stress response in Synechococcus elongatus PCC 7942 Hickman, Jason W.; Kotovic, Kimberly M.; Miller, Cameron Algal Research, Vol. 2, Issue 2 https://doi.org/10.1016/j.algal.2013.01.008	journal	March 2013
Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size Kirst, Henning; Formighieri, Cinzia; Melis, Anastasios Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1837, Issue 10 https://doi.org/10.1016/j.bbabio.2014.07.009	journal	October 2014
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 152, Issue 5, p. 1173-1183 https://doi.org/10.1016/j.cell.2013.02.022	journal	February 2013
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes Gilbert, Luke A.; Larson, Matthew H.; Morsut, Leonardo Cell, Vol. 154, Issue 2, p. 442-451 https://doi.org/10.1016/j.cell.2013.06.044	journal	July 2013
Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway Cameron, Jeffrey C.; Wilson, Steven C.; Bernstein, Susan L. Cell, Vol. 155, Issue 5 https://doi.org/10.1016/j.cell.2013.10.044	journal	November 2013
Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes Copeland, Matthew F.; Politz, Mark C.; Pfleger, Brian F. Current Opinion in Biotechnology, Vol. 29 https://doi.org/10.1016/j.copbio.2014.02.010	journal	October 2014
Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli Pitera, Douglas J.; Paddon, Chris J.; Newman, Jack D. Metabolic Engineering, Vol. 9, Issue 2 https://doi.org/10.1016/j.ymben.2006.11.002	journal	March 2007
Synthetic biology: New strategies for directing design Lynch, Sean A.; Gill, Ryan T. Metabolic Engineering, Vol. 14, Issue 3 https://doi.org/10.1016/j.ymben.2011.12.007	journal	May 2012
Synthetic biology and the development of tools for metabolic engineering Keasling, Jay D. Metabolic Engineering, Vol. 14, Issue 3 https://doi.org/10.1016/j.ymben.2012.01.004	journal	May 2012
Enhancing fatty acid production by the expression of the regulatory transcription factor FadR Zhang, Fuzhong; Ouellet, Mario; Batth, Tanveer S. Metabolic Engineering, Vol. 14, Issue 6 https://doi.org/10.1016/j.ymben.2012.08.009	journal	November 2012
Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi) Cleto, Sara; Jensen, Jaide VK; Wendisch, Volker F. ACS Synthetic Biology, Vol. 5, Issue 5 https://doi.org/10.1021/acssynbio.5b00216	journal	February 2016
Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002 Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E. ACS Synthetic Biology, Vol. 4, Issue 5 https://doi.org/10.1021/sb500260k	journal	September 2014
Engineered CRISPR-Cas9 nucleases with altered PAM specificities Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q. Nature, Vol. 523, Issue 7561 https://doi.org/10.1038/nature14592	journal	June 2015
Automated design of synthetic ribosome binding sites to control protein expression Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A. Nature Biotechnology, Vol. 27, Issue 10, p. 946-950 https://doi.org/10.1038/nbt.1568	journal	October 2009
Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes Pfleger, Brian F.; Pitera, Douglas J.; Smolke, Christina D. Nature Biotechnology, Vol. 24, Issue 8 https://doi.org/10.1038/nbt1226	journal	July 2006
Genomic mining of prokaryotic repressors for orthogonal logic gates Stanton, Brynne C.; Nielsen, Alec A. K.; Tamsir, Alvin Nature Chemical Biology, Vol. 10, Issue 2 https://doi.org/10.1038/nchembio.1411	journal	December 2013
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Characterization of 582 natural and synthetic terminators and quantification of their design constraints Chen, Ying-Ja; Liu, Peng; Nielsen, Alec A. K. Nature Methods, Vol. 10, Issue 7 https://doi.org/10.1038/nmeth.2515	journal	June 2013
CRISPR interference (CRISPRi) for sequence-specific control of gene expression Larson, Matthew H.; Gilbert, Luke A.; Wang, Xiaowo Nature Protocols, Vol. 8, Issue 11 https://doi.org/10.1038/nprot.2013.132	journal	October 2013
Synthetic biology to access and expand nature's chemical diversity Smanski, Michael J.; Zhou, Hui; Claesen, Jan Nature Reviews Microbiology, Vol. 14, Issue 3 https://doi.org/10.1038/nrmicro.2015.24	journal	February 2016
Fuelling the future: microbial engineering for the production of sustainable biofuels Liao, James C.; Mi, Luo; Pontrelli, Sammy Nature Reviews Microbiology, Vol. 14, Issue 5 https://doi.org/10.1038/nrmicro.2016.32	journal	March 2016
Protein-based organelles in bacteria: carboxysomes and related microcompartments Yeates, Todd O.; Kerfeld, Cheryl A.; Heinhorst, Sabine Nature Reviews Microbiology, Vol. 6, Issue 9 https://doi.org/10.1038/nrmicro1913	journal	August 2008
Coordination of microbial metabolism Chubukov, Victor; Gerosa, Luca; Kochanowski, Karl Nature Reviews Microbiology, Vol. 12, Issue 5 https://doi.org/10.1038/nrmicro3238	journal	March 2014
An updated evolutionary classification of CRISPR–Cas systems Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S. Nature Reviews Microbiology, Vol. 13, Issue 11, p. 722-736 https://doi.org/10.1038/nrmicro3569	journal	September 2015
Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli Wu, Junjun; Du, Guocheng; Chen, Jian Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep13477	journal	September 2015
ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY Ludwig, Martha; Sültemeyer, Dieter; Price, G. Dean Journal of Phycology, Vol. 36, Issue 6 https://doi.org/10.1046/j.1529-8817.2000.00028.x	journal	December 2000
Tuning genetic control through promoter engineering Alper, H.; Fischer, C.; Nevoigt, E. Proceedings of the National Academy of Sciences, Vol. 102, Issue 36, p. 12678-12683 https://doi.org/10.1073/pnas.0504604102	journal	August 2005
PipX, the coactivator of NtcA, is a global regulator in cyanobacteria Espinosa, Javier; Rodríguez-Mateos, Francisco; Salinas, Paloma Proceedings of the National Academy of Sciences, Vol. 111, Issue 23 https://doi.org/10.1073/pnas.1404097111	journal	May 2014
Improving fatty acids production by engineering dynamic pathway regulation and metabolic control Xu, P.; Li, L.; Zhang, F. Proceedings of the National Academy of Sciences, Vol. 111, Issue 31 https://doi.org/10.1073/pnas.1406401111	journal	July 2014
Nitrogen Induction of Sugar Catabolic Gene Expression in Synechocystis sp. PCC 6803 Osanai, Takashi; Imamura, Sousuke; Asayama, Munehiko DNA Research, Vol. 13, Issue 5 https://doi.org/10.1093/dnares/dsl010	journal	January 2006
OPTIMIZER: a web server for optimizing the codon usage of DNA sequences Puigbo, P.; Guzman, E.; Romeu, A. Nucleic Acids Research, Vol. 35, Issue Web Server https://doi.org/10.1093/nar/gkm219	journal	May 2007
Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites Espah Borujeni, Amin; Channarasappa, Anirudh S.; Salis, Howard M. Nucleic Acids Research, Vol. 42, Issue 4 https://doi.org/10.1093/nar/gkt1139	journal	November 2013
Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system Bikard, David; Jiang, Wenyan; Samai, Poulami Nucleic Acids Research, Vol. 41, Issue 15 https://doi.org/10.1093/nar/gkt520	journal	June 2013
Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803 Grundel, M.; Scheunemann, R.; Lockau, W. Microbiology, Vol. 158, Issue Pt_12 https://doi.org/10.1099/mic.0.062950-0	journal	October 2012
Sequence determinants of improved CRISPR sgRNA design Xu, Han; Xiao, Tengfei; Chen, Chen-Hao Genome Research, Vol. 25, Issue 8 https://doi.org/10.1101/gr.191452.115	journal	June 2015
Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity Lea-Smith, David J.; Bombelli, Paolo; Dennis, John S. Plant Physiology, Vol. 165, Issue 2 https://doi.org/10.1104/pp.114.237206	journal	April 2014
Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA Espinosa, Javier; Forchhammer, Karl; Burillo, Sergio Molecular Microbiology, Vol. 61, Issue 2 https://doi.org/10.1111/j.1365-2958.2006.05231.x	journal	July 2006
Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803 Krasikov, Vladimir; Aguirre von Wobeser, Eneas; Dekker, Henk L. Physiologia Plantarum, Vol. 145, Issue 3 https://doi.org/10.1111/j.1399-3054.2012.01585.x	journal	March 2012
The Production of Hydrogen Peroxide by Blue-Green Algae: a Survey Stevens, S. E.; Patterson, C. O. Pat; Myers, Jack Journal of Phycology, Vol. 9, Issue 4 https://doi.org/10.1111/j.1529-8817.1973.tb04116.x	journal	December 1973
Phycobilisomes Supply Excitations to Both Photosystems in a Megacomplex in Cyanobacteria Liu, H.; Zhang, H.; Niedzwiedzki, D. M. Science, Vol. 342, Issue 6162 https://doi.org/10.1126/science.1242321	journal	November 2013
Rerouting Carbon Flux To Enhance Photosynthetic Productivity Ducat, Daniel C.; Avelar-Rivas, J. Abraham; Way, Jeffrey C. Applied and Environmental Microbiology, Vol. 78, Issue 8 https://doi.org/10.1128/AEM.07901-11	journal	February 2012
Effects of Modified Phycobilin Biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002 Alvey, R. M.; Biswas, A.; Schluchter, W. M. Journal of Bacteriology, Vol. 193, Issue 7 https://doi.org/10.1128/JB.01392-10	journal	February 2011
Nitrogen Control in Cyanobacteria Herrero, A.; Muro-Pastor, A. M.; Flores, E. Journal of Bacteriology, Vol. 183, Issue 2 https://doi.org/10.1128/JB.183.2.411-425.2001	journal	January 2001
The phycobilisome, a light-harvesting complex responsive to environmental conditions. Grossman, A. R.; Schaefer, M. R.; Chiang, G. G. Microbiological Reviews, Vol. 57, Issue 3 https://doi.org/10.1128/MR.57.3.725-749.1993	journal	January 1993
The phycobilisome, a light-harvesting complex responsive to environmental conditions. Grossman, A. R.; Schaefer, M. R.; Chiang, G. G. Microbiological Reviews, Vol. 57, Issue 3 https://doi.org/10.1128/mr.57.3.725-749.1993	journal	January 1993
Development of bio-based fine chemical production through synthetic bioengineering Hara, Kiyotaka Y.; Araki, Michihiro; Okai, Naoko Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/s12934-014-0173-5	journal	December 2014
An Organic Acid Based Counter Selection System for Cyanobacteria Begemann, Matthew B.; Zess, Erin K.; Walters, Eric M. PLoS ONE, Vol. 8, Issue 10 https://doi.org/10.1371/journal.pone.0076594	journal	October 2013
Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria Farasat, I.; Kushwaha, M.; Collens, J. Molecular Systems Biology, Vol. 10, Issue 6, p. 731-731 https://doi.org/10.15252/msb.20134955	journal	June 2014
Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002 Davies, Fiona K.; Work, Victoria H.; Beliaev, Alexander S. Frontiers in Bioengineering and Biotechnology, Vol. 2 https://doi.org/10.3389/fbioe.2014.00021	journal	June 2014
Transcription Profiling of the Model Cyanobacterium Synechococcus sp. Strain PCC 7002 by Next-Gen (SOLiD™) Sequencing of cDNA Ludwig, Marcus; Bryant, Donald A. Frontiers in Microbiology, Vol. 2 https://doi.org/10.3389/fmicb.2011.00041	journal	January 2011
Acclimation of the Global Transcriptome of the Cyanobacterium Synechococcus sp. Strain PCC 7002 to Nutrient Limitations and Different Nitrogen Sources Ludwig, Marcus; Bryant, Donald A. Frontiers in Microbiology, Vol. 3 https://doi.org/10.3389/fmicb.2012.00145	journal	January 2012

Cited By (11)

Metabolic pathway rewiring in engineered cyanobacteria for solar-to-chemical and solar-to-fuel production from CO₂ Woo, Han Min Bioengineered, Vol. 9, Issue 1 https://doi.org/10.1080/21655979.2017.1317572	journal	May 2017
Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference Libby, Ashley R. G.; Joy, David A.; So, Po-Lin bioRxiv https://doi.org/10.1101/252189	journal	January 2018
Progress and challenges in engineering cyanobacteria as chassis for light‐driven biotechnology Hitchcock, Andrew; Hunter, C. Neil; Canniffe, Daniel P. Microbial Biotechnology, Vol. 13, Issue 2 https://doi.org/10.1111/1751-7915.13526	journal	August 2019
CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942 Huang, Chun-Hung; Shen, Claire R.; Li, Hung Microbial Cell Factories, Vol. 15, Issue 1 https://doi.org/10.1186/s12934-016-0595-3	journal	November 2016
Insight into Crispr System in Eukaryotic Microalgae, a Review Bilal, I. G.; Butnariu, M.; Bilal, E. EurasianUnionScientists, Vol. 3, Issue 59 https://doi.org/10.31618/esu.2413-9335.2019.3.59.37-48	journal	January 2019
New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering Santos-Merino, María; Singh, Amit K.; Ducat, Daniel C. Frontiers in Bioengineering and Biotechnology, Vol. 7 https://doi.org/10.3389/fbioe.2019.00033	journal	February 2019
Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942 Kim, Wook Jin; Lee, Sun-Mi; Um, Youngsoon Frontiers in Plant Science, Vol. 8 https://doi.org/10.3389/fpls.2017.00293	journal	March 2017
Enhanced Production of D-Lactate in Cyanobacteria by Re-Routing Photosynthetic Cyclic and Pseudo-Cyclic Electron Flow Selão, Tiago Toscano; Jebarani, Jasmin; Ismail, Nurul Aina Frontiers in Plant Science, Vol. 10 https://doi.org/10.3389/fpls.2019.01700	journal	January 2020
Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects Al-Haj, Lamya; Lui, Yuen; Abed, Raeid Life, Vol. 6, Issue 4 https://doi.org/10.3390/life6040042	journal	November 2016
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology Gale, Grant A. R.; Schiavon Osorio, Alejandra A.; Mills, Lauren A. Microorganisms, Vol. 7, Issue 10 https://doi.org/10.3390/microorganisms7100409	journal	September 2019
Additional file 2 of Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli Li, Ying; Xian, He; Xu, Ya figshare https://doi.org/10.6084/m9.figshare.13698050	dataset	January 2021

Similar Records

Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973

Journal Article · Wed Dec 11 19:00:00 EST 2019 · ACS Synthetic Biology · OSTI ID:1800965

Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

Journal Article · Thu Mar 26 20:00:00 EDT 2015 · Life, 5(2):1127-1140 · OSTI ID:1182919

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
CRISPRi
carboxysome
chemical production
cyanobacteria
lactate
phycobilisome
synthetic biology
tunable

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Citation Formats

References (58)

Cited By (11)

Similar Records

Related Subjects