Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)
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 expr...
| Main Authors: | , , , |
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| Other Authors: | , , |
| Format: | Article |
| Language: | English |
| Published: |
American Chemical Society,
2018-02-20T14:53:38Z.
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| Subjects: | |
| Online Access: | Get fulltext |
| Summary: | 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 National Institutes of Health (U.S.) (DP2 OD008435) National Institutes of Health (U.S.) (P50 GM098792) United States. Office of Naval Research (N00014-13-1-0424) National Science Foundation (U.S.) (MCB1350625) Wuxi NewWay Biotech Ltd. (Jiangsu Province International Collaboration Grant BZ2014014) |
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