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 »
- Authors:
-
- University of Wisconsin, Madison, WI (United States)
- 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:
- 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. Fri .
"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},
journal = {Metabolic Engineering},
number = ,
volume = 38,
place = {United States},
year = {Fri Jul 29 00:00:00 EDT 2016},
month = {Fri Jul 29 00:00:00 EDT 2016}
}
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