Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)
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Lu_Corynebacterium.pdf
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Author(s)
Wendisch, Volker F.
Cleto, Sara
Jensen, Jaide
Lu, Timothy K
Date Issued
February 2016
Journal
ACS Synthetic Biology
Publisher
American Chemical Society
Citation
Cleto, Sara, Jaide VK Jensen, Volker F. Wendisch, and Timothy K. Lu. “Corynebacterium glutamicumMetabolic Engineering with CRISPR Interference (CRISPRi).” ACS Synthetic Biology 5, no. 5 (May 20, 2016): 375–385.
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Author's final manuscript
Abstract
Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.
Keywords: amino acid; C. glutamicum; CRISPRi; metabolic engineering; sgRNA/dCas9
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Massachusetts Institute of Technology. Synthetic Biology Center
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1021/acssynbio.5b00216
