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. 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. 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.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States; Microbiology Doctoral Training Program, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, United States.
authors.forename: Gina C
authors.initials: GC
authors.lastname: Gordon
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States; Department of Environmental Chemistry and Technology, University of Wisconsin-Madison, 660 N Park St, Madison, WI 53706, United States.
authors.forename: Travis C
authors.initials: TC
authors.lastname: Korosh
authors.affiliation: Departmentof Chemistry and Biochemistry, University of Colorado-Boulder, 596 UCB, Boulder, CO 80309, United States; Renewable and Sustainable Energy Institute, University of Colorado-Boulder, 027 UCB, Boulder, CO 80309, United States.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Cameron
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Markley
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States; Microbiology Doctoral Training Program, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, United States.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Begemann
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States; Microbiology Doctoral Training Program, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, United States. Electronic address: pfleger@engr.wisc.edu.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003198:Computer Simulation; D000072669:Gene Editing / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000941:ethics; D008954:Models, Biological; D015398:Signal Transduction; D013045:Species Specificity; D013297:Streptococcus pyogenes / Q000235:genetics*; D046940:Synechococcus / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D015534:Trans-Activators
keywords: CRISPRi; Carboxysome; Chemical production; Cyanobacteria; Lactate; Phycobilisome; Synthetic biology; Tunable