Abstract
BackgroundThe construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host.ResultsTo demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (l-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of l-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer).ConclusionsSingle or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum.
Highlights
The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering
We report the development of a twoplasmid CRISPR interference (CRISPRi) and the detail studies with single guide RNAs for single or double repression of target genes in C. glutamicum wild-type (Fig. 1a)
Construction of the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) interference for C. glutamicum To construct the two-plasmid system of the CRISPRi (Table 1 and Fig. 1b), the CoryneBrick vector system [11] was used for expression of the dCas9 gene encoding deactivated Cas9 nuclease under pTetA promoter, and a high-copy plasmid was modified to express a single guide RNA under the constitutive promoter, based on CRISPRi of the Qi’ lab [12]
Summary
The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host. A CRISPRi system for the metabolic engineering of C. glutamicum has been developed to repress single genes [9] for improvement of l-lysine and l-glutamate production, and the CRISPRi system has been applied to regulate multiple genes for shikimic acid production [10]. We report the development of a twoplasmid CRISPRi and the detail studies with single guide RNAs for single or double repression of target genes in C. glutamicum wild-type (Fig. 1a). The CRISPRi vectors could be a cost-effective and time-saving metabolic engineering tool and will promote for researchers to investigate controlling gene expression for biochemical production in C. glutamicum
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