Design and characterization of a synthetic minimal promoter for heterocyst-specific expression in filamentous cyanobacteria

PLoS One. 2018 Sep 11;13(9):e0203898. doi: 10.1371/journal.pone.0203898. eCollection 2018.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anabaena / genetics
  • Bacterial Proteins / genetics
  • Biotechnology
  • Consensus Sequence
  • Cyanobacteria / genetics*
  • Cyanobacteria / growth & development
  • Cyanobacteria / metabolism
  • DNA, Bacterial / genetics
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Genes, Bacterial
  • Genes, Reporter
  • Genes, Synthetic*
  • Luminescent Proteins / genetics
  • Nitrogen Fixation / genetics
  • Nostoc / genetics
  • Nostoc / growth & development
  • Nostoc / metabolism
  • Promoter Regions, Genetic*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Luminescent Proteins
  • yellow fluorescent protein, Bacteria

Grant support

This work was supported by NordForsk (https://www.nordforsk.org/en), NCoE program “NordAqua” (project # 82845), and the Swedish Energy Agency (https://www.energimyndigheten.se/en/),(project # 11674-5) to KS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.