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Available online at www.sciencedirect.com

ScienceDirect

European Journal of Protistology 75 (2020) 125720

ioactive molecules from protists: Perspectives in biotechnology

driana Vallesia,∗, Sandra Pucciarellia,∗, Federico Buonannob, Angelo Fontanac,arco Mangiagallid

School of Biosciences and Veterinary Medicine, Università degli Studi di Camerino, Camerino (MC), ItalyLaboratory of Protistology and Biology Education, Department of E.C.H.T. Università degli Studi di Macerata, Macerata, ItalyBio-Organic Chemistry Unit, CNR-Institute of Biomolecular Chemistry, Pozzuoli, Napoli, ItalyDepartment of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

eceived 23 February 2020; received in revised form 11 May 2020; accepted 15 May 2020vailable online 4 June 2020

bstract

For hundreds of years, mankind has benefited from the natural metabolic processes of microorganisms to obtain basic productsuch as fermented foods and alcoholic beverages. More recently, microorganisms have been exploited for the production ofntibiotics, vitamins and enzymes to be used in medicine and chemical industries. Additionally, several modern drugs, includinghose for cancer therapy, are natural products or their derivatives. Protists are a still underexplored source of natural productsotentially of interest for biotechnological and biomedical applications. This paper focuses on some examples of bioactiveolecules from protists and associated bacteria and their possible use in biotechnology.

2020 Elsevier GmbH. All rights reserved.

eywords: Bioactive molecules from protists; Natural products; Cold-adapted enzymes; Ice binding proteins; Climacostol; Secondaryetabolites

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ntroduction

Protists are fundamental components of microbial commu-ities in every aquatic and terrestrial habitat worldwide, fromhe ocean depths to the highest mountain peaks. Inhabitingll environments, including extreme ones characterized byow or high temperatures (Oliverio et al. 2018; Valbonesi anduporini 1993), high salinity (Harding et al. 2017), and low origh pH values (Ong’ondo et al. 2013), protists have evolved

n amazing biodiversity and unique mechanisms of adapta-ion which involve synthesis of specific molecules required tohrive in these environments. In addition, protists use chemi-

∗Corresponding authors.E-mail addresses: [email protected] (A. Vallesi),

[email protected] (S. Pucciarelli).

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al signals to face predators and capture prey (Buonanno et al.013), communicate within the same species or among differ-nt species (Frenkel et al. 2014; Ianora et al. 2011; Luporinit al. 2016a; Luporini et al. 2016b), and mediate interactionsetween symbiotic organisms (Amin et al. 2012; Pohnertt al. 2007; Roy et al. 2013). This large array of productsffers a new and unexplored source of bioactive compoundsith potential biotechnological applications in industrial sec-

ors related to food, cosmetics and human health (Assunçãot al. 2017; Peyrat et al. 2019; Singh et al. 2017; Sun et al.015).The demand for bioactive molecules with desirable

roperties is constantly growing, and the development of

odern technological methods for molecular purification

nd the increasing availability of genome data provide newpproaches for the discovery of natural products and the study

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A. Vallesi, S. Pucciarelli, F. Buonanno et al. /

f their applications. The main classes of bioactive moleculesre primary metabolites (e.g. amino acids, enzymes, lipids,itamins etc.) essential for the growth of microorganismsSingh et al. 2017; Sun et al. 2015), and secondary metabo-ites, which are small organic compounds produced duringhe stationary phase of growth, with antimicrobial and cyto-oxic activities (Demain 1999).

This review reports recent advancements in this specificeld of protistological research discussed at the symposiumBioactive molecules from protists: perspectives in biotech-ology” during the VIII European Congress of Protistology –SOP joint meeting (Rome, 2019). Natural products isolatedrom diatoms, dinoflagellates and ciliates have shown greatotential in various fields. The cold-adapted enzymes fromntarctic ciliates and the ice binding proteins synthesized by

heir associated bacteria display peculiar physical-chemicaleatures that make them exploitable for industrial purposes.n diatoms, specific classes of lipids act as signals of celleath and growth termination; manipulation of lipid biosyn-hesis can thus be used to control diatom growth for biomassroduction, making these microalgae a promising font of bio-uels, food raw materials and other value-added products.econdary metabolites synthesized by numerous aquatic cil-

ates for chemical defense and/or offence in predator–preynteractions are now emerging as potential chemotherapeuticgents with immune-modulatory and anti-cancer activi-ies.

nzymes and ice-binding proteins fromrotists and their associated bacteria

Enzymes play a key role in biotechnological and indus-rial applications. In some cases, industrial processes presentarsh conditions (e.g. low or high temperatures, acidic orasic pHs, the presence of organic solvents) or requireigh or low substrate specificity (Sarmiento et al. 2015).n this context, enzymes from extremophilic organisms arearticularly attractive; for instance, enzymes active at lowemperatures find application in detergency, food indus-ry and molecular biology (Collins and Margesin 2019;

angiagalli et al. 2020; Sarmiento et al. 2015), while ther-ophilic enzymes find application in food and in pulp and

aper industries, as well as in molecular biology (Sarmientot al. 2015). The enzymes obtained from protists and char-cterized to date, while few in number, indicate the greatotential of these microorganisms as powerful source for newiomolecules.

The characterization of enzymes from “extreme” protistss interesting not only for industrial applications, but alsoor the study of the mechanisms of molecular evolution anddaptation to the environment. For instance, studies carriedut on cold-active �-amylase and superoxide dismutase from

he psychrophilic Antarctic ciliate Euplotes focardii providednsights into the relationship between activity at low temper-ture and stability to heat (Pischedda et al. 2018; Yang et al.

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an Journal of Protistology 75 (2020) 125720

013). In particular, E. focardii superoxide dismutases arective at 4 ◦C and retain residual activity even after incubationt 50 ◦C (Fig. 1A) (Pischedda et al. 2018).

Ice binding proteins (IBPs) represent another class ofolecules with application in food industries and in cry-

preservation of biological materials (Kim et al. 2017;angiagalli et al. 2020). These proteins prevent cell freezing

y depressing the freezing point of water below the meltingoint and inhibit ice recrystallization by binding to the surfacef ice crystals (Fig. 1) (Bar Dolev et al. 2016; Davies 2014;ance et al. 2019; Voets 2017). Different IBPs have been iden-

ified from psychrophilic protists including Antarctic diatomsBayer-Giraldi et al. 2010; Xiao et al. 2014), unicellularlgae (Raymond 2014; Raymond and Morgan-Kiss 2013;aymond and Remias 2019) and unicellular fungi (Arai et al.019; Cheng et al. 2016; Kondo et al. 2012). A new IBPEfcIBP) has been identified and extensively characterizedrom the metagenomic analysis of the bacterial consortiumssociated to the Antarctic ciliate E. focardii (Mangiagallit al. 2017; Mangiagalli et al. 2018; Pucciarelli et al. 2015).iochemical characterization of recombinant EfcIBP showed

hat this protein is one of the best-performing IBPs describedo date in inhibiting the growth of large harmful ice crys-als (Mangiagalli et al. 2017). This finding indicates thatven protist-associated bacteria may prove valuable in theearch for new molecules to be used in industrial applica-ions.

iomass, carotenoids and fatty acids fromicroalgae

Microalgae are widespread photosynthetic microorgan-sms inhabiting marine and freshwater environments, and aref great interest in biotechnology as a source of biomass andioactive molecules (e.g. carotenoids, fatty acids and antiox-dants) used as additives in food, cosmetic and animal feedndustries (Assunção et al. 2017; Borowitzka 2013; Ganglt al. 2015).

Diatoms have pivotal roles in biological and geochemi-al marine cycles (Armbrust 2009; Benoiston et al. 2017).ecause these protists gain their energy from photosynthe-

is and use dissolved CO2 as a building block (Reinfeldert al. 2004), they have developed very efficient mecha-isms for maintaining high metabolic plasticity that allowshem to balance growth, production of functional compoundsnd accumulation of storage products (Adelfi et al. 2019;utignano et al. 2006; D’Ippolito et al. 2004; Gerecht et al.011; Lamari et al. 2013). Diatom blooms are at the very basisf the marine food web and they are primary producers ofssential molecules such as eicosapentaenoic acid (EPA) thatre transferred though the web to higher organisms, includ-ng human beings (Brett et al. 2009; Larson et al. 2013). The

atural ability of diatoms to produce large biomass has alsorompted interest in the commercial exploitation of theseroducts (Katiyar et al. 2017; Lebeau and Robert 2003).

A. Vallesi, S. Pucciarelli, F. Buonanno et al. / European Journal of Protistology 75 (2020) 125720 3

Fig. 1. Proteins from E. focardii and its bacterial consortium. (A) Superoxide dismutases (SODs) from E. focardii. The three different SODsi tivity a ◦

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dentified in the Antarctic ciliate E. focardii show remarkable acischedda et al. (2018). (B) Binding of EfcIBP to ice crystals ind

nhibiting the growth of the large ones.

In general, large-scale cultures of microalgae started morehan 50 years ago in open ponds. These cultivations do notompete in land and space with terrestrial crops, and couldontribute to mitigate the negative effects of carbon dioxidend other pollutants on the environment. The main studiesn this field have traditionally focused on engineering solu-ions to reduce capital and operating expenditures, as well asosts of downstream processes such as harvesting and dewa-ering (Assunção et al. 2017; Ugwu et al. 2008). However,ecent researches have been directed to both the selection ofesilient species and the development of biological methodso improve production yields (Klok et al. 2014; Rodolfi et al.009; Vaezi 2015). From this point of view, the biodiversityffered by the estimated 100,000 diatom species living in thearth’s waters can be a wealth of new strains and source of dif-

erent target products (D’Ippolito et al. 2015). Lately, studiesn the cosmopolitan diatom species Thalassiosira weissflodgiave demonstrated the feasibility of year-round outdoor cul-

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t 4 C, coupled with unusual heat stability. Figure adapted fromhe freezing point depression and stabilized the small ice crystals

ivation, with no need to control ambient temperature andight irradiance (Vella et al. 2019). Biomass (4 g/m2 d) and oil0.5 g/m2 d) productivity of this cultivation was lower thanhat of indoor reference cultures (12 g/m2 d and 4 g/m2 d,espectively), but significant improvements can be achievedy adjustments to culture conditions such as nutrient qual-ty and availability, salinity, pH, and dissolved oxygen andarbon dioxide.

As in other microalgae, nutrient limitation triggers producthanges in diatoms (i.e., reduced levels of silicon increase oilccumulation) (Botte et al. 2018), but the response seems toary from one species to another. Molecular and biochemicalpproaches are other possible tools for controlling productynthesis. In the last decade, studies have begun to outlinehe eco-physiological processes that regulate diatom growth,

ncluding a number of lipid-based mechanisms involvedn both cell death programs and allelopathy (Bidle 2016;ontana et al. 2007; Franklin et al. 2006). These mecha-

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isms can be employed to direct synthesis of specific productst high rates or to control biomass productivity (Bacellarendes and Vermelho 2013; Hamilton et al. 2014). For

nstance, sterol sulfates have been shown to be implied inhysiological cell death in the marine diatom Skeletonemaostatum. The biochemical inhibition of their synthesis has aeneficial effect on cells with a significant increase of the cellumber and the amount of produced biomass in comparisono control cultures under the same conditions (Gallo et al.017, 2018, 2020).Among products extracted from microalgae, �-carotene

s synthesized in large amounts from the halophilic algaunaliella salina (Borowitzka 2013), and astaxanthin, onef the most powerful antioxidants, from the freshwater algaaematococcus pluvialis (Ambati et al. 2014). Species such

s D. salina, T. weissflogii and Cyclotella cryptica are alsonown for their ability to store triacyl-glycerides, which rep-esent 60–80% of the total lipid content (D’Ippolito et al.015; Scott et al. 2010). This finding suggests that microal-ae could be exploited for production of biodiesel, which isroving to be a viable alternative to fossil fuels.

econdary metabolites and drug discoveryrom aquatic protists

Aquatic organisms live in a particularly competitive envi-onment, and for this reason have evolved a great numberf adaptive strategies, many of which are devoted to manag-ng predator–prey interactions. While some organisms havepecific offensive/defensive morphological structures, othersynthesize toxic secondary metabolites which are typicallysed for chemical defense and/or offence. Secondary metabo-ites are small organic molecules whose molecular weightsually is less than 1000 Da; they perform different physio-ogical or ecological functions in the organism that produceshem, and show a considerable structural diversity and “priv-leged scaffolds”, with molecular characteristics adapted tohe specific interaction with cellular targets. The amazinghemical and structural diversity of these bioactive moleculess unequalled by any synthetic library, providing the basis forhe design of a variety of new effective drugs (Carugo andraetta 2019; Catalani et al. 2016).The marine environment hosts a wide variety of organisms

n terms of physiological characteristics and adaptability.ince the late sixties (Carroll et al. 2020), marine organ-

sms have been the subject of several bioprospecting studiesor the identification of new therapeutic agents (Newmannd Cragg 2020). Currently, 13 approved drugs are basedn marine natural products and more than 20 moleculesre in the pharmaceutical pipeline (Altmann 2017; Carroll

t al. 2020; Liang et al. 2019; Newman and Cragg 2016).he majority of these compounds are used as anticancergents and are produced from or were originally identi-ed in invertebrates (mostly sponges and molluscs), while

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rotists are still largely unexplored, mainly because it isifficult to have sufficient material for bioassay-guided purifi-ation and structure elucidation. In effect, the process of drugiscovery involves pharmacological tests with raw extractsollowed by isolation and characterization of the componentsesponsible for the activity. In the specific case of marineroducts, the presence of high salt concentrations in cellxtracts requires appropriate sample preparation and frac-ionation procedures (Broach and Thorner 1996; Cobice et al.015; Cutignano et al. 2015; Hughes et al. 2011; Zhang et al.016), and large amounts of starting material are usuallyeeded to purify the active molecules. Although it is diffi-ult to obtain these amounts from protists, there are somexceptions.

Dinoflagellates and diatoms produce several differentiotoxins that are traditionally grouped on the basis on theirffects upon humans: paralytic shellfish poisoning (PSP),eurotoxic shellfish poisoning (NSP), diarrheic shellfishoisoning (DSP) and amnesic shellfish poisoning (ASP),zaspiracid poisoning (AZP), and ciguatera fish poisoningCFP) (Carroll et al. 2020; Fu et al. 2017; Kitchen et al.018; Rasmussen et al. 2016; Sakai and Swanson 2014;an Dolah 2000; Wang 2008). Many of these compoundsre used as powerful tools for research in cell biology andave been suggested for several pharmaceutical applicationselated to blockage of ion channels and transmission of elec-rical impulses in neurons and muscle cells (Assunção et al.017; Gerwick and Moore 2012; Harvey 2008; Newman andragg 2004; Romano et al. 2017).Gambierol, gymnodimine, yessotoxin and other biotoxins

ave immunotherapeutic activity and have been proposed asmmunosuppressants in dysfunctional immune system dis-ases, including neurodegenerative diseases (Assunção et al.017). The mechanisms of action of these molecules areather diverse and include reversible down-regulation of the

cell receptor complex in T lymphocytes by yessotoxin andkadaic acid (Martín-López et al. 2011), potassium channel-ependent inhibition of resting T lymphocytes (Rubiolo et al.015), or activation of proinflammatory pathways in humanacrophages (Crinelli et al. 2012). In addition, the sym-

iotic dinoflagellates of the genus Amphidinium producewo large families of molecules known as amphidinols andmphidinolides that have antibacterial and antifungal prop-rties (Kobayashi 2008; Kobayashi and Tsuda 2004).

Immunomodulatory activity is the main characteristicf Sulfavant A (Fig. 2A), a synthetic derivative of �-ulfoquinovosides of the diatom T. weissflodgii (Manzo et al.017). Alpha-sulfoquinovosides are major components ofhloroplast membranes in this diatom and are common inany photosynthetic organisms. Sulfavant induces the mat-

ration of dendritic cells with up-regulation of phenotypicarkers in a dose-dependent manner and enhances cell sur-

ace expression of CD83, a critical marker for in vivo primingf naive T cells. Used in vivo in B16F10 mice, Sulfavant

increases antigen-specific immune protection and signifi-antly delays the onset and development of melanoma tumor

A. Vallesi, S. Pucciarelli, F. Buonanno et al. / European Journal of Protistology 75 (2020) 125720 5

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Fig. 2. Chemical structures of Sulfavants. (

hen the molecule is associated with the human peptidep10025-33. The boost of the immune response by Sulfa-ant has thus suggested its use as vaccine adjuvant; sinceulfavant has a mechanism of action independent from Toll-ike Receptors, it is potentially much less toxic than otherdjuvants. Recently, other analogs with activity 1000 timesore potent than Sulfavant A have been reported (Fig. 2B)

Manzo et al. 2019). In addition to antigen and adjuvant,accine formulation can include several additives such astabilizers, preservatives, solubilizers and surfactants. Themphiphilic molecule of Sulfavant A and the attitude toorm self-aggregation make this compound able to form sta-le suspensions without the addition of other ingredientsManzo et al. 2019). These chemo-physical properties ofulfavant A and analogs affect the immunological efficiencynd can offer a technical advantage in the vaccine formula-ion.

The toxin climacostol and its derivates exemplify howhe combination of chemistry and biology has allowedhe production of new molecules of pharmacological inter-st. Climacostol (5-[(2Z)-non-2-en-1-yl] benzene-1,3-diol) isynthesized by the heterotrich freshwater ciliate Climacosto-

um virens for defense against unicellular or multicellularredators (Buonanno et al. 2013; Miyake et al. 2003).his molecule exhibits antimicrobial and cytotoxic activities

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favant A (1); (B) Sulfavant S (2) and R (3).

gainst pathogenic bacteria, fungi and protozoa (Fig. 3A)Buonanno and Ortenzi 2010; Buonanno and Ortenzi 2018;etrelli et al. 2012). Moreover, it is able to inhibit the prolifer-tion of human and rodent cell lines through the induction ofNA damage and apoptosis (Buonanno et al. 2008; Perrotta

t al. 2016; Quassinti et al. 2013).Climacostol has been available as a synthetic compound

ince 1999 (Masaki et al. 1999; Masaki et al. 2004), andore recently it has been chemically synthesized as a pure

ompound in the natural and most bioactive Z-configurationFiorini et al. 2010), allowing researchers to design androduce different synthetic analogs (Buonanno et al. 2019;atalani et al. 2019). The addition of a methyl group to

he aromatic ring of Climacostol enhances its antimicrobialnd cytotoxicity activities, while the addition of a hydroxylroup in the same position induces apoptosis in protozoaFig. 3B) (Buonanno et al. 2019). On the other hand, theddition of a methoxymethyl ether protective group to theromatic ring of Climacostol resulted in the synthesis of arodrug (Fig. 3B) which can be activated by acidic con-itions (pH < 7). This prodrug appears to be particularlyseful for delivering the non-toxic protected Climacostol to

cidic pathological multicellular or unicellular targets suchs tumor cells and parasites, where it shifts to the active form.ested in vitro in melanoma cells and in vivo in Drosophila

6 A. Vallesi, S. Pucciarelli, F. Buonanno et al. / European Journal of Protistology 75 (2020) 125720

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ig. 3. Structure and activity of Climacostol, the toxic secondaryf Climacostol. (B) The MOM-protected Climacostol (MOMO) slimacostol and their functions.

elanogaster fruit flies, this modified molecule is able toeduce viability by induction of apoptosis (Catalani et al.019).

onclusion and perspective

Due to their biodiversity and ubiquity, protists are a goldine for the discovery of new bioactive molecules which are

till almost unknown. In many cases, the paucity of informa-ion about natural products and secondary metabolites from

rotists depends mainly on the lack of adequate techniquesor obtaining the required biomass for molecular isolationnd identification. Along with technological improvements,

moo

olite produced by Climacostomum virens. (A) Natural functions active Climacostol if exposed to pH < 7, and the two analogs of

enetic and biochemical manipulations seem to be the nextteps to develop the biotechnological use of natural prod-cts. In addition, the growing number of protist genomesnd DNA sequences deposited in public databases will pavehe way for finding new molecules by a genome miningpproach.

We do not know to what extent molecules from protists wille used in biotechnological applications as biologically activepharmaceutical) and functional (nutraceutical and cosmetic)ompounds. In any case, the discovery of new molecules

ay contribute to better understanding of the mechanisms

f adaptation to different environments, including extremenes.

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uthor contributions

All authors contributed equally to this work.

cknowledgments

AF thanks the colleagues from the Bioorganic Chem-stry Unit, namely Carmela Gallo, Emiliano Manzo, Giuliana’Ippolito, Genoveffa Nuzzo and Raffaele De Palma.

FB thanks: Elisabetta Catalani, Davide Cervia, Francescaroietti Serafini, Simona Picchietti, Anna Maria Fausto of

he Department for Innovation in Biological, Agro-food andorest systems (DIBAF), Università degli Studi della Tus-ia, Viterbo, Italy; Simone Giorgi, Gabriele Lupidi, Federicoittorio Rossi, Enrico Marcantoni of the School of Sci-nces and Technologies, Section of Chemistry, Università diamerino; Claudio Ortenzi of the Laboratory of Protistologynd Biology Education, Department of E.C.H.T. Universitài Macerata, Macerata, Italy.SP and MM thank the H2020-MSCA-RISE program and

he Progetto Nazionale di Ricerche in Antartide (PEA), AVhe support of the University of Camerino (Fondo di Ateneoer la Ricerca 2018). The authors would also like to thankheila Beatty for editing the English usage in the manuscript.

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8 Europe

C

C

C

C

D

D

D

D

F

F

F

F

F

G

G

G

G

G

G

H

H

H

H

I

K

K

K

K

K

K

K

L

L

L

L


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