uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Design and characterization of a synthetic minimal promoter for heterocyst-specific expression in filamentous cyanobacteria
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial chemistry)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial chemistry)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Univ Nottingham, Ctr Biomol Sci, Univ Pk, Nottingham, England.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial chemistry)ORCID iD: 0000-0001-6993-8476
2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 9, article id e0203898Article in journal (Refereed) Published
Abstract [en]

Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.

Place, publisher, year, edition, pages
2018. Vol. 13, no 9, article id e0203898
National Category
Biochemistry and Molecular Biology Genetics
Research subject
Chemistry with specialization in Microbial Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-343828DOI: 10.1371/journal.pone.0203898ISI: 000444355500056PubMedID: 30204806OAI: oai:DiVA.org:uu-343828DiVA, id: diva2:1186865
Funder
Swedish Energy Agency, 11674-5NordForsk, 82845Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-11-14Bibliographically approved
In thesis
1. Development of robust cyanobacterial strains for biotechnological applications: Stress tolerance and cell-specific expression in heterocyst-forming cyanobacteria
Open this publication in new window or tab >>Development of robust cyanobacterial strains for biotechnological applications: Stress tolerance and cell-specific expression in heterocyst-forming cyanobacteria
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Synthetic biology tools can be used to exploit potential applications of cyanobacteria, promising photosynthetic hosts for production of fuels and chemicals. Specific genetic tools are needed for the development of robust cyanobacterial strains for bioengineering. The key work presented in this thesis is a characterization and design of bioengineering tools for the heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133. This multicellular cyanobacterium may express several oxidative stress-managing systems, including five Dps proteins. Two of these Dps proteins, NpDps2 and NpDps5, are involved in the tolerance against oxidative stress that induced by H2O2 or high light intensities. The capacity of NpDps2 and NpDps5 to further enhance oxidative stress tolerance, was confirmed by homologous overexpression and a constitutive strong promoter in N. punctiforme. The results show the potential of Dps proteins as tools to create robust cyanobacterial cells with improved stress tolerance. This work also establishes a Dps-mediated link among light tolerance, H2O2 scavenging, and iron homeostasis, and provides evidence on the non-redundant role of multiple Dps in multicellular cyanobacteria. To address the lack of well-defined promoters in cyanobacteria, a minimal synthetic promoter, SynDIF, was designed for heterocyst-specific expression. Promoters with 5’TCCGGA, the DIF motif, at the -35 region, have been identified to give heterocyst-specific transcription. To identify promoter elements, critical for cell-specificity, DIF promoter sequences from Anabaena PCC 7120 were used in a consensus sequence approach. The importance of the consensus regions for cell-specificity was investigated with promoter-eyfp reporters. This result provides new insights to the details of DIF promoters, which suggest that the DIF-motif, only together with the consensus or a native DIF promoter -10 region, are sufficient for heterocyst-specificity. Therefore, the DIF-35 region was (i) not independent of other promoter elements, and (ii) not sufficient for heterocyst-specific expression. Besides, the strength of the synthetic promoter was improved by including the upstream element from the native heterocyst specific promoter PNsiR1. Moreover, the SynDIF promoter is the shortest promoter ever reported to provide heterocyst-specific expression, indicating the potential of introducing this promoter in future biotechnological applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 80
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1641
Keywords
Cyanobacteria,  Dps protein,  heterocysts,  Nostoc,  oxidative stress,  cell specific promoter, ROS tolerance, synthetic biology tools transcriptional regulation.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-343829 (URN)978-91-513-0259-1 (ISBN)
Public defence
2018-04-20, 10134, Polhemsalen, Lägerhyddsvägen 1, Ångström Laboratory, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2018-03-26 Created: 2018-03-01 Last updated: 2018-04-24

Open Access in DiVA

fulltext(5233 kB)4 downloads
File information
File name FULLTEXT01.pdfFile size 5233 kBChecksum SHA-512
526773fa9c1136257bf421ce8148297ede584c4c1766248e07ce4801be46b9f324da9172206fae8be957b83c14863bf81a91ff9822dac07ea7a7153afdd3c91e
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Authority records BETA

Wegelius, AdamLi, XinStensjö, Karin

Search in DiVA

By author/editor
Wegelius, AdamLi, XinStensjö, Karin
By organisation
Molecular Biomimetics
In the same journal
PLoS ONE
Biochemistry and Molecular BiologyGenetics

Search outside of DiVA

GoogleGoogle Scholar
Total: 4 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 53 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf