4 years ago

Novel promoters for constitutive and inducible expression of transgenes in the diatom Phaeodactylum tricornutum under varied nitrate availability

Stefan Leu, Aliza Zarka, Inna Khozin-Goldberg, Zachor Adler-Agnon (Shemesh), Sammy Boussiba


Recent developments in the genetic engineering of biotechnologically important microalgae—among them the diatom Phaeodactylum tricornutum—promise rapid progress in strain improvements for producing various valuable commodities. Nitrogen (N) depletion and starvation are commonly employed strategies to induce or enhance the formation of secondary carotenoids and storage lipids in microalgal biotechnology. The manipulation of gene expression for improving or rerouting the metabolic pathways requires the development of suitable stage-specific promoters to drive gene expression under desired conditions. Here, we describe the isolation, cloning, and characterization of novel P. tricornutum promoters that are either constitutively active in the presence or absence of nitrate as the sole N source, or upregulated under N starvation. An enhanced green fluorescent protein (eGFP), fused to a plasma membrane-anchoring sequence at the C terminus, was expressed under the control of several endogenous promoters, which were chosen based on previously reported gene expression levels under nitrate-limited or deprived conditions. Among the four promoters tested in this work, the ammonium transporter gene (AMT, PHATRDRAFT_27877) promoter strongly induced eGFP accumulation under N starvation, and the purine permease gene (PUP, PHATRDRAFT_52619) promoter strongly and constitutively drove eGFP accumulation, regardless of nitrate availability. The Act2 (actin-like 2, PHATRDRAFT_29136) and previously characterized DGAT1 gene promoters had relatively weak constitutive and late-inducible expression, respectively, under N starvation. The fucoxanthin-chlorophyll a/c-binding protein gene (fcpA) promoter used as a control was downregulated under N starvation. A comparison of quantitative real-time PCR gene expression data with western blot results suggested that controlled transcriptional regulation is important for efficient transgenic protein accumulation under N starvation. The results are of interest for the expression of transgenes in P. tricornutum for engineering oil production capacity and accumulating recombinant proteins.

Publisher URL: https://link.springer.com/article/10.1007/s10811-017-1335-8

DOI: 10.1007/s10811-017-1335-8

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.