skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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 ofmore » 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.« less

Authors:
 [1];  [1];  [2];  [1];  [1];  [1]
  1. University of Wisconsin, Madison, WI (United States)
  2. University of Colorado, Boulder, CO (United States)
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); National Institutes of Health (NIH)
OSTI Identifier:
1424612
Alternate Identifier(s):
OSTI ID: 1436836
Grant/Contract Number:  
SC0010329; EFRI-1240268; 5 T32 GM08349
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 38; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; CRISPRi; tunable; cyanobacteria; lactate; chemical production; carboxysome; phycobilisome; synthetic biology

Citation Formats

Gordon, Gina C., Korosh, Travis C., Cameron, Jeffrey C., Markley, Andrew L., Begemann, Matthew B., and Pfleger, Brian F. CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. United States: N. p., 2016. Web. doi:10.1016/j.ymben.2016.07.007.
Gordon, Gina C., Korosh, Travis C., Cameron, Jeffrey C., Markley, Andrew L., Begemann, Matthew B., & Pfleger, Brian F. CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. United States. https://doi.org/10.1016/j.ymben.2016.07.007
Gordon, Gina C., Korosh, Travis C., Cameron, Jeffrey C., Markley, Andrew L., Begemann, Matthew B., and Pfleger, Brian F. 2016. "CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002". United States. https://doi.org/10.1016/j.ymben.2016.07.007. https://www.osti.gov/servlets/purl/1424612.
@article{osti_1424612,
title = {CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002},
author = {Gordon, Gina C. and Korosh, Travis C. and Cameron, Jeffrey C. and Markley, Andrew L. and Begemann, Matthew B. and Pfleger, Brian F.},
abstractNote = {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.},
doi = {10.1016/j.ymben.2016.07.007},
url = {https://www.osti.gov/biblio/1424612}, journal = {Metabolic Engineering},
issn = {1096-7176},
number = ,
volume = 38,
place = {United States},
year = {Fri Jul 29 00:00:00 EDT 2016},
month = {Fri Jul 29 00:00:00 EDT 2016}
}

Journal Article:

Citation Metrics:
Cited by: 123 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Tuning genetic control through promoter engineering
journal, August 2005


Effects of Modified Phycobilin Biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002
journal, February 2011


An Organic Acid Based Counter Selection System for Cyanobacteria
journal, October 2013


Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
journal, June 2013


Phycobilisomes of the Cyanobacterium Synechococcus SP. PCC 7002: Structure, Function, Assembly, and Expression
book, January 1990


Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway
journal, November 2013


Characterization of 582 natural and synthetic terminators and quantification of their design constraints
journal, June 2013


Coordination of microbial metabolism
journal, March 2014


Insights into the industrial growth of cyanobacteria from a model of the carbon-concentrating mechanism
journal, January 2014


Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)
journal, February 2016


Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes
journal, October 2014


Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002
journal, June 2014


Rerouting Carbon Flux To Enhance Photosynthetic Productivity
journal, February 2012


Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites
journal, November 2013


PipX, the coactivator of NtcA, is a global regulator in cyanobacteria
journal, May 2014


Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria
journal, June 2014


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009


CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
journal, July 2013


Development of bio-based fine chemical production through synthetic bioengineering
journal, December 2014


Nitrogen Control in Cyanobacteria
journal, January 2001


Glycogen synthesis is a required component of the nitrogen stress response in Synechococcus elongatus PCC 7942
journal, March 2013


Site-directed mutagenesis by overlap extension using the polymerase chain reaction
journal, April 1989


Synthetic biology and the development of tools for metabolic engineering
journal, May 2012


Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size
journal, October 2014


Engineered CRISPR-Cas9 nucleases with altered PAM specificities
journal, June 2015


Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803
journal, March 2012


CRISPR interference (CRISPRi) for sequence-specific control of gene expression
journal, October 2013


Fuelling the future: microbial engineering for the production of sustainable biofuels
journal, March 2016


Phycobilisomes Supply Excitations to Both Photosystems in a Megacomplex in Cyanobacteria
journal, November 2013


Synthetic biology: New strategies for directing design
journal, May 2012


An updated evolutionary classification of CRISPR–Cas systems
journal, September 2015


Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002
journal, September 2014


Regulation of ammonium assimilation in cyanobacteria
journal, June 2003


Metabolic design for cyanobacterial chemical synthesis
journal, April 2014


Nitrogen Induction of Sugar Catabolic Gene Expression in Synechocystis sp. PCC 6803
journal, January 2006


Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes
journal, July 2006


Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli
journal, March 2007


OPTIMIZER: a web server for optimizing the codon usage of DNA sequences
journal, May 2007


Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
journal, February 2013


A single-point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor
journal, January 2011


Automated design of synthetic ribosome binding sites to control protein expression
journal, October 2009


Synthetic biology to access and expand nature's chemical diversity
journal, February 2016


Genomic mining of prokaryotic repressors for orthogonal logic gates
journal, December 2013


The Production of Hydrogen Peroxide by Blue-Green Algae: a Survey
journal, December 1973


Sequence determinants of improved CRISPR sgRNA design
journal, June 2015


Improving fatty acids production by engineering dynamic pathway regulation and metabolic control
journal, July 2014


Protein-based organelles in bacteria: carboxysomes and related microcompartments
journal, August 2008


Enhancing fatty acid production by the expression of the regulatory transcription factor FadR
journal, November 2012


Works referencing / citing this record:

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference
journal, January 2018


Progress and challenges in engineering cyanobacteria as chassis for light‐driven biotechnology
journal, August 2019


CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
journal, November 2016


Insight into Crispr System in Eukaryotic Microalgae, a Review
journal, January 2019


New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering
journal, February 2019


Enhanced Production of D-Lactate in Cyanobacteria by Re-Routing Photosynthetic Cyclic and Pseudo-Cyclic Electron Flow
journal, January 2020


Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects
journal, November 2016


Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology
journal, September 2019