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

Title: 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. ^[1]; Cameron, Jeffrey C. ^[2]; Markley, Andrew L. ^[1]; Begemann, Matthew B. ^[1]; Pfleger, Brian F. ^[1]

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.

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

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

Grant/Contract Number:: SC0010329; EFRI-1240268; 5 T32 GM08349

OSTI ID:: 1424612

Alternate ID(s):: OSTI ID: 1436836

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

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 123 works

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

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

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