PolyUnsaturated Fatty acids in Antarctic diatoms: metabolic biodiversity and impact of Global WARming

Acronym
PUFA-WAR
Code
PNRA19_00105, line B
Anno
2024
Research area
Life science
Specific research topic
The study of cold-adapted enzymes involved in the PUFA pathways in Antarctic diatoms
Region of interest
Antarctica, Rothera Station
Project website
PI
Daniela Giordano
PI establishment
National Research Council (CNR)- Institute of Biosciences and BioResources (IBBR)
Institutional website
https://www.ibbr.cnr.it/ibbr/info/people/daniela-giordano
Other institutions and subjects involved
Prof. Stefano Bruno, University of Parma (UNIPR); Prof. Melody Clark, British Antarctic Survey (BAS)
Consistency of the research team
2 PhD (females), 1 post-doc (female), 9 researchers (5 females and 4 males)
Project status
In progress
Main stations used
Basi Straniere
The project

Diatoms represent one of the major groups of photosynthetic algae and account for more than half of the total primary production at the base of the global Food Web. They are also primary producers of polyunsaturated fatty acids (PUFAs). Particularly, in the Antarctic species, elevated levels of PUFAs counter the effects of elevated hydrostatic pressures and low temperatures in the modulation of membrane fluidity. The PUFAs’ composition of diatoms varies considerably within taxa and can be affected by temperature, light and nutrient conditions. As PUFAs produced by microalgae are essential to all metazoans, understanding the influence of environmental changes on the PUFAs biosynthetic pathways in diatoms is a crucial piece of information to predict the possible outcomes of Global Warming on the entire ecosystem of the Southern Ocean. In this framework, the project aims to study the genetic and metabolic diversity of cold-adapted enzymes involved in the PUFA pathways in Antarctic diatoms by i) identifying the genes encoding for these enzymes and ii) studying their transcription levels and the resulting lipidome when grown under stress conditions. The team is composed of three Research Units (RU): RU1-CNR, headed by Dr. Daniela Giordano of the National Research Council (CNR), RU2-UNIPR, headed by Prof. Stefano Bruno of the University of Parma (UNIPR) and a foreign partner unit, headed by Prof. Melody Clark of the British Antarctic Survey (BAS), who has expertise in the study of the environmental genomics of Antarctic organisms. Her team has already isolated and successfully cultivated Antarctic diatom strains collected during campaigns at Rothera Station, Antarctica. This proposal is within the framework of the international SCAR biology Programme “Antarctic Thresholds-Ecosystem Resilience and Adaptation (AnT-ERA)”, to which both BAS and CNR take part.

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  • Motivation, importance of research

    Diatoms are key organisms in marine food webs for the production of long-chain polyunsaturated fatty acids (LC-PUFAs) and metazoans – which cannot produce PUFAs but need them for a variety of cellular functions –  have to acquire them from dietary sources. The most common LC-PUFAs produced by marine microalgae are the omega-3 fatty acids eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Particularly, EPA and DHA ascend the food chain to accumulate in marine fish and in higher trophic levels (Walsh et al 2016). There is also a large species-specific variability in EPA and DHA content.
    In diatoms, PUFAs have an important role in maintaining cell membrane fluidity and are essential for photosynthesis, playing a key role in the function of the thylakoid membrane. Indeed, one of the key adaptive strategies of cold-adapted microalgae to the extreme habitat of Antarctica is the elevation of PUFA levels in membranes to increase their fluidity and to maintain cell metabolism at lower water temperatures. Therefore, PUFAs may have a a more critical role for the diatoms of the polar region in comparison to those of other environments, although little is known on their PUFA metabolism.

    Within this framework, understanding the influence of environmental changes on the biosynthetic pathway involved in the expression of PUFAs in Antarctic microalgae is an essential piece of information to predict the possible effects of Global Warming on the entire ecosystem of the Southern Ocean. To date, some physiological studies have been conducted to evaluate the content and productivity of PUFAs in Antarctic species, but only a few studies have focused on the identification of the enzymes involved in PUFA biosynthesis. 
     

    Objectives of the proposal

    The objectives of this project are:


    i) selection of representative psychrophilic diatom strains among those already isolated by the foreign partner (Prof. Melody Clark);
     

    ii) identification of the genes encoding for the cold-adapted enzymes involved in the EPA and DHA pathways in selected Antarctic diatoms to study their genetic metabolic biodiversity; in the case of uncertainties on substrates specificity of the protein that have emerged, they will be expressed recombinantly in Escherichia coli or in other organisms and their reactivity will be assayed by mass spectrometry approaches;
     

    iii) investigate the transcription levels of mRNAs encoding for these enzymes in the same strains grown under different conditions of temperature and light exposure and correlate the results with the different lipidomic profiles.
     

    Activities carried out and results achieved

    Activities for this two-year project are: 

    1) Identification and characterization of genes encoding for the cold-adapted enzymes involved in the PUFA pathways in the Antarctic diatom Fragilariopsis cylindrus to study its genetic metabolism: amplification, sequencing, analysis, and alignment;

    2) Cloning of genes encoding for the cold-adapted enzymes involved in the PUFA pathways in expression vectors and overexpression in Escherichia coli and/or Pichia pastoris and evaluation of the reaction catalyzed by the recombinant enzymes;

    3) Culturing of Fragilariopsis cylindrus under different stress conditions (temperature, light)

    a) Evaluation of its expression levels of mRNAs encoding for these enzymes by qRT-PCR;

    b) Evaluation of the lipidome profile of the selected diatom subjected to stress conditions in comparison to cultures grown at optimal conditions 

    c) Evaluation of the epigenetics profiles of the genes encoding desaturares and elongases involved in the PUFA biosynthesis 

    Expected results:

    • -Improved knowledge on the genetic metabolic diversity of Antarctic diatom strains
    • -Identification and analysis of genes involved in the elongation and desaturation of PUFA
    • -Understanding of the impact of global warming mediated by temperature and light on the expression of PUFAs in diatoms of the Southern Ocean.
    Products

    R. Giaccari, D. Giordano, M. Clark, M. Davey, N. Thomas, A. Thomson, F. Vacondio, C. Verde, S. Bruno. Nutraceuticals from the cold: impact of global warming on the polyunsaturated fatty acid biosynthesis in the Antarctic diatom Fragilariopsis cylindrus. Poster presented at FEBS 2024, Milan, Italy