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Multiple Gene Repression in Cyanobacteria Using CRISPRi
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8317-1654
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-2430-2682
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-1899-7649
2016 (English)In: ACS Photonics, ISSN 2186-2311, E-ISSN 2161-5063, Vol. 5, no 3, 207-212 p.Article in journal (Refereed) PublishedText
Abstract [en]

We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II. CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50-95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016. Vol. 5, no 3, 207-212 p.
Keyword [en]
Strain Pcc 6803, Sequence-Specific Control, Synechocystis Sp Pcc6803, Escherichia-Coli, Carbon-Dioxide, Light, Expression, Dehydrogenase, Tolerance, Aldehyde
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-185366DOI: 10.1021/acssynbio.5b00264ISI: 000372672500003PubMedID: 26689101ScopusID: 2-s2.0-84961794371OAI: oai:DiVA.org:kth-185366DiVA: diva2:920613
Funder
Swedish Research Council Formas, 213-2011-1655Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Foundation for Strategic Research , RBP14-0013
Note

QC 20160418

Available from: 2016-04-18 Created: 2016-04-18 Last updated: 2016-04-22Bibliographically approved
In thesis
1. Metabolic engineering strategies to increase n-butanol production from cyanobacteria
Open this publication in new window or tab >>Metabolic engineering strategies to increase n-butanol production from cyanobacteria
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n­-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance.

In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days.

In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold.

In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis.

Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor.

Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 79 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:4
Keyword
cyanobacteria, metabolic engineering, biofuels, butanol, synthetic scaffold, CRISPRi, solvent tolerance
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-185548 (URN)978-91-7595-927-6 (ISBN)
Public defence
2016-05-27, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council FormasKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Available from: 2016-04-22 Created: 2016-04-21 Last updated: 2016-04-28Bibliographically approved

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