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CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

BACKGROUND: 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 Cas9 (dCas9) and single g...

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Autores principales: Huang, Chun-Hung, Shen, Claire R., Li, Hung, Sung, Li-Yu, Wu, Meng-Ying, Hu, Yu-Chen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111286/
https://www.ncbi.nlm.nih.gov/pubmed/27846887
http://dx.doi.org/10.1186/s12934-016-0595-3
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author Huang, Chun-Hung
Shen, Claire R.
Li, Hung
Sung, Li-Yu
Wu, Meng-Ying
Hu, Yu-Chen
author_facet Huang, Chun-Hung
Shen, Claire R.
Li, Hung
Sung, Li-Yu
Wu, Meng-Ying
Hu, Yu-Chen
author_sort Huang, Chun-Hung
collection PubMed
description BACKGROUND: 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 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. RESULTS: To 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. CONCLUSIONS: These 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 MATERIAL: The online version of this article (doi:10.1186/s12934-016-0595-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-51112862016-11-21 CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942 Huang, Chun-Hung Shen, Claire R. Li, Hung Sung, Li-Yu Wu, Meng-Ying Hu, Yu-Chen Microb Cell Fact Research BACKGROUND: 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 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. RESULTS: To 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. CONCLUSIONS: These 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 MATERIAL: The online version of this article (doi:10.1186/s12934-016-0595-3) contains supplementary material, which is available to authorized users. BioMed Central 2016-11-15 /pmc/articles/PMC5111286/ /pubmed/27846887 http://dx.doi.org/10.1186/s12934-016-0595-3 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Huang, Chun-Hung
Shen, Claire R.
Li, Hung
Sung, Li-Yu
Wu, Meng-Ying
Hu, Yu-Chen
CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title_full CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title_fullStr CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title_full_unstemmed CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title_short CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
title_sort crispr interference (crispri) for gene regulation and succinate production in cyanobacterium s. elongatus pcc 7942
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111286/
https://www.ncbi.nlm.nih.gov/pubmed/27846887
http://dx.doi.org/10.1186/s12934-016-0595-3
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