期刊
METABOLIC ENGINEERING
卷 38, 期 -, 页码 170-179出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2016.07.007
关键词
CRISPRi; Tunable; Cyanobacteria; Lactate; Chemical production; Carboxysome; Phycobilisome; Synthetic biology
资金
- US Department of Energy [DE-SC0010329]
- National Science Foundation [EFRI-1240268]
- William F. Vilas Trust
- NIH Biotechnology Training Fellowships (NIGMS) [5 T32 GM08349]
- U.S. Department of Energy (DOE) [DE-SC0010329] Funding Source: U.S. Department of Energy (DOE)
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [1215871] Funding Source: National Science Foundation
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. (C)) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
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