Abstract
BackgroundCyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells.ResultsTo employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58–0.63 mg/L.ConclusionsThese data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0595-3) contains supplementary material, which is available to authorized users.
Highlights
Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells
We constructed pSdCas9-CY’ with the expression cassette comprised of chloramphenicol resistance gene (CmR), dCas9 under Psmt, and eyfp under PconII, which was flanked by homology arms targeting the neutral site I (NSI) site (Fig. 1a)
The single guide RNA (sgRNA) were designed to target the eyfp cassette at the non-template strand of promoter (P1) or coding regions near the transcription start site (NT1) or near the middle of gene (NT2) so that the fluorescence intensity served as the indicator of gene repression
Summary
Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas (dCas9) and single guide RNA (sgRNA) to repress sequencespecific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various pro‐ caryotic cells. Many cyanobacterial strains are amenable to natural transformation and homologous recombination for gene manipulation. Thanks to these attributes, genetically engineered cyanobacteria have drawn increasing attention as a chassis for the production of biofuels and bio-derived chemicals [2].
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