Green Microalgae Strain Improvement for the Production of Sterols and Squalene

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Article Green Microalgae Strain Improvement for the Production of Sterols and Squalene

Supakorn Potijun 1,2 , Suparat Jaingam 1,2, Nuttha Sanevas 3 , Srunya Vajrodaya 3 and Anchalee Sirikhachornkit 1,2,*

1 Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; [email protected] (S.P.); [email protected] (S.J.) 2 Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU), Kasetsart University, Bangkok 10900, Thailand 3 Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; [email protected] (N.S.); [email protected] (S.V.) * Correspondence: [email protected]; Tel.: +66-2562-5444; Fax: +66-2579-5528

Abstract: Sterols and squalene are essential biomolecules required for the homeostasis of eukaryotic membrane permeability and fluidity. Both compounds have beneficial effects on human health. As the current sources of sterols and squalene are plant and shark oils, microalgae are suggested as more sustainable sources. Nonetheless, the high costs of production and processing still hinder the commercialization of algal cultivation. Strain improvement for higher product yield and tolerance to harsh environments is an attractive way to reduce costs. Being an intermediate in sterol synthesis, squalene is converted to squalene epoxide by squalene epoxidase. This step is inhibited by terbinafine, a commonly used antifungal drug. In yeasts, some terbinafine-resistant strains overproduced sterols, but similar microalgae strains have not been reported. Mutants that exhibit greater tolerance to Citation: Potijun, S.; Jaingam, S.; terbinafine might accumulate increased sterols and squalene content, along with the ability to tolerate Sanevas, N.; Vajrodaya, S.; Sirikhachornkit, A. Green Microalgae the drug and other stresses, which are beneficial for outdoor cultivation. To explore this possibility, Strain Improvement for the terbinafine-resistant mutants were isolated in the model green microalga Chlamydomonas reinhardtii Production of Sterols and Squalene. using UV mutagenesis. Three mutants were identified and all of them exhibited approximately 50 Plants 2021, 10, 1673. https:// percent overproduction of sterols. Under terbinafine treatment, one of the mutants also accumulated doi.org/10.3390/plants10081673 around 50 percent higher levels of squalene. The higher accumulation of pigments and triacylglycerol were also observed. Along with resistance to terbinafine, this mutant also exhibited higher resistance Academic Editors: Sousuke Imamura to oxidative stress. Altogether, resistance to terbinafine can be used to screen for strains with increased and Imran Pancha levels of sterols or squalene in green microalgae without growth compromise.

Received: 29 May 2021 Keywords: biodiesel; Chlamydomonas; squalene; sterol; terbinaﬁne Accepted: 11 August 2021 Published: 13 August 2021

Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Sterols belong to a class of isoprenoid lipids found in the eukaryotic membrane. They iations. perform crucial roles in maintaining membrane fluidity and permeability [1]. In animals, cholesterol is the major sterol that is essential for various biological processes. In fungi, ergosterol is the major type of sterol [2,3]. Sterols in plants or phytosterols perform similar functions as does cholesterol in terms of physiological functions and structure. Phytosterols have been clinically proven to be beneficial for human consumption [4]. They possess Copyright: © 2021 by the authors. anti-cancer, anti-inflammatory, immunomodulatory and neuromodulatory properties [5–7]. Licensee MDPI, Basel, Switzerland. This article is an open access article The consumption of phytosterols have been shown to lower blood LDL-cholesterol levels distributed under the terms and and decrease the chance of developing a number of diseases [8,9]. The current source conditions of the Creative Commons of phytosterols is from plant oil. However, due to the rising demand for phytosterols, Attribution (CC BY) license (https:// microalgae have been suggested as an alternative source, as algal oils contain equal or creativecommons.org/licenses/by/ greater amounts of sterols compared to plant oils [9,10]. Moreover, some microalgae also 4.0/).

Plants 2021, 10, 1673. https://doi.org/10.3390/plants10081673 https://www.mdpi.com/journal/plants PlantsPlants2021 2021, ,10 10,, 1673 x FOR PEER REVIEW 22 of of 13 13

synthesizealso synthesize certain certain sterols sterols such assuch 7-dehydroporiferasterol, as 7-dehydroporiferasterol, that are that not are present not present in plants, in whichplants, has which been has shown been toshown possess to possess outstanding outstanding anti-inflammatory anti-inflammatory activities activities [11]. [11]. TheThe synthesis of of sterols sterols starts starts with with isopen isopentenyltenyl diphosphate diphosphate (IPP), (IPP), a five-carbon a five-carbon com- compoundpound that thatacts actsas a asbuilding a building block block for all for isoprenoids all isoprenoids (Figure (Figure 1). In1). plants In plants and and algae, algae, IPP IPPcan canbe derived be derived from from both boththe mevalonate the mevalonate (MVA) (MVA) pathway, pathway, and the and non-mevalonate the non-mevalonate or the ormethyl-D-erythritol the methyl-D-erythritol 4-phosphate 4-phosphate (MEP) pathway (MEP) pathway [12]. In green [12]. Inalgae, green however, algae, however,the MVA thehad MVA been hadlost, beenleaving lost, the leaving MEP thepathway MEP pathwayas the sole as pathway the sole pathwayfor isoprenoid for isoprenoid synthesis synthesis[13,14]. The [13 first,14]. step The of firststerol step synthesis of sterol in green synthesis algae in is greenthe production algae is the of squalene. production Squa- of squalene.lene is widely Squalene used in is widelythe pharmaceutical used in the and pharmaceutical cosmetics industries and cosmetics for its industriesability to hold for itsmoisture, ability toits holdantioxidant moisture, and its anticancer antioxidant activities, and anticancer and its protective activities, effect and against its protective cardio- effectvascular against diseases cardiovascular [15,16]. It is diseases also commonly [15,16]. Itused is also as an commonly ingredient used in vaccine as an ingredient adjuvants in[17]. vaccine Currently, adjuvants the major [17]. Currently,sources of squalene the major are sources plant ofoil squalene and shark are liver plant oil. oil Due and to shark these liverunsustainable oil. Due to sources, these unsustainable microorganisms sources, includin microorganismsg microalgae have including also been microalgae explored have for alsothe commercial been explored production for the commercial of squalene production [18]. of squalene [18].

FigureFigure 1.1. SimpliﬁedSimplified pathwayspathways forfor sterol,sterol, pigment,pigment, andand triacylglyceroltriacylglycerol synthesissynthesis (G3P;(G3P; glycerol-3-phosphate;glycerol-3-phosphate; GGPP:GGPP: geranylgeranyl pyrophosphate; IPP: isopentenyl diphosphate; LPA: lysophosphatidic acid; MEP: 2-C-methyl-d-erythritol geranylgeranyl pyrophosphate; IPP: isopentenyl diphosphate; LPA: lysophosphatidic acid; MEP: 2-C-methyl-d-erythritol 4-phosphate; TAG: triacylglycerol). 4-phosphate; TAG: triacylglycerol).

TheThe cultivationcultivation ofof microalgaemicroalgae hashas beenbeen widelywidely suggestedsuggested asas aa wayway ofof utilizingutilizing lightlight energyenergy fromfrom sunlightsunlight forfor thethe productionproduction ofof highhigh valuevaluecompounds, compounds, andand simultaneouslysimultaneously reducingreducing atmosphericatmospheric carboncarbon dioxidedioxide viavia photosynthesisphotosynthesis [ 19[19].]. AsAs algaealgae cancan utilizeutilize organicorganic mattermatter from industrial industrial water water for for growth, growth, the the production production of natural of natural products products could could be com- be combinedbined with with wastewater wastewater treatment. treatment. Nevertheless, Nevertheless, several several limitations limitations such as such the ascost the of cost cul- oftivation, cultivation, cell harvesting, cell harvesting, and compound and compound extraction extraction still exist. still exist. Outdoor Outdoor cultivation cultivation is also is alsofaced faced with with several several obstacles obstacles such such as high as highlight lightintensity intensity and fluctuating and ﬂuctuating temperature, temperature, both bothof which of which cause cause oxidative oxidative stress stress to the to cell, the cell,lowering lowering the biomass the biomass yield. yield. Contamination Contamination and andinvasion invasion by other by other algal algal species species are are also also common common [20,21]. [20,21]. Therefore, Therefore, algal strainsstrains withwith higherhigher resistanceresistance toto abioticabiotic stress,stress, oror beingbeing ableable toto growgrow inin conditionsconditions whichwhich othersothers cannotcannot

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tolerate would be favorable. Improving selected strains to increase yield of high value Plants 2021, 10, 1673 products is another direct approach to boost production efficiency [22]. 3 of 13 Terbinafine is an antifungal drug that prevents the accumulation of sterols, leading to cell death [23]. It inhibits the enzyme squalene epoxidase, which converts squalene to squalenetolerate epoxide would be (Figure favorable. 1). Terbinafine Improving selectedwas reported strains to increaseinteract yieldwith ofthe high enzyme, value pre- ventingproducts the isnatural another substrate direct approach from binding to boost productionto the enzyme’s efficiency active [22]. site [24]. It is possible that greaterTerbinafine tolerance is an to antifungal this drug drug might that be prevents a result the of accumulation greater ability of sterols, to generate leading more substrateto cell deathmolecules [23]. Itor inhibits better activity the enzyme of the squalene enzyme epoxidase, to bind whichwith its converts substrate, squalene squalene, whichto squalene ultimately epoxide leads (Figure to overproduction1). Terbinafine of was sterols. reported Other to interact than resistance with the enzyme,to this drug, resistantpreventing strains the might natural have substrate a higher from tolerance binding to to the other enzyme’s stresses active that site microalgae [24]. It is possible might face that greater tolerance to this drug might be a result of greater ability to generate more sub- in an outdoor cultivation setting. To explore this idea, UV mutagenesis was employed to strate molecules or better activity of the enzyme to bind with its substrate, squalene, which generateultimately a mutant leads to population overproduction of the of sterols.model Othergreen than microalga resistance Chlamydomonas to this drug, resistant reinhardtii. Threestrains terbinafine-resistant might have a higher mutants tolerance were to other successfully stresses that isolated. microalgae Physiological might face characteri- in an zationoutdoor of the cultivation mutants setting.was performed To explore and this discussed. idea, UV mutagenesis was employed to generate a mutant population of the model green microalga Chlamydomonas reinhardtii. Three 2. Resultsterbinafine-resistant and Discussion mutants were successfully isolated. Physiological characterization of the mutants was performed and discussed. 2.1. Mutant Isolation and the Determination of Sterols, Squalene, and Pigment Content 2.The Results inhibition and Discussion of intermediate steps in the sterol biosynthesis decreased the accumu- lation2.1. of Mutant sterol Isolation in plants and theand Determination algae [25–28]. of Sterols, Cells Squalene,that are andable Pigment to resist Content inhibitors in the sterol biosynthesisThe inhibition might of intermediate then have steps an upregula in the steroltion biosynthesis of a certain decreased step in the accumula-pathway, or a changetion ofin sterolthe activity in plants of and certain algae enzymes, [25–28]. Cells leading that areto sterol able to accumulation. resist inhibitors A in UV-mutagen- the sterol izedbiosynthesis mutant population might then of have 1920 an colo upregulationnies was generated of a certain in step the in green the pathway, alga C. orreinhardii a change. They werein tested the activity on plates of certain containing enzymes, different leading co toncentrations sterol accumulation. of terbinafine. A UV-mutagenized Three terbinafine- mutant population of 1920 colonies was generated in the green alga C. reinhardii. They sensitive strains were isolated and characterized in our previously published work [29]. were tested on plates containing different concentrations of terbinafine. Three terbinafine- Fromsensitive this same strains mutant were isolatedpopulation, and characterized a total of three in our mutants previously were published confirmed work for [29their]. re- sistanceFrom thisto the same drug mutant (Figure population, 2). They a totalwere of named three mutants terbinafine-resistant were confirmed mutants, for their resis- tfr1, tfr2, andtance tfr3. toTo the study drug the (Figure effects2). They of terbinafine were named resist terbinafine-resistantance on the sterol mutants, content,tfr1 ,thetfr2 ,predomi- and nanttfr3 sterols. To study in Chlamydomonas the effects of terbinafine, ergosterol resistance and 7-dehydroporiferasterol, on the sterol content, the were predominant analyzed us- ingsterols the GC-MS in Chlamydomonas method. All, ergosterol three mutants and 7-dehydroporiferasterol, exhibited an increase of were approximately analyzed using 50% in thethe level GC-MS of both method. sterols All compared three mutants to the exhibited WT (Figure an increase 3A). ofIn approximately the presence 50%of terbinafine, in the whichlevel targets of both the sterols squalene compared epoxidase to the WT enzyme, (Figure 3squaleneA). In the accumulation presence of terbinafine, can be observed which in targets the squalene epoxidase enzyme, squalene accumulation can be observed in this this alga. The tfr1 mutant accumulated 50% higher squalene level, whereas tfr2 and tfr3 alga. The tfr1 mutant accumulated 50% higher squalene level, whereas tfr2 and tfr3 showed showed50% lower 50% squalenelower squalene level compared level compared to that of theto that WT (Figureof the WT3B). (Figure 3B).

Figure 2. Growth phenotype of wild type (WT) and tfr mutants of C. reinhardtii. Serial dilutions of cells were spotted onto Figurethe 2. agarGrowth medium phenotype without of terbinaﬁne wild type (left (WT)) and and with tfr 1 mutants mM terbinaﬁne of C. reinhardtii (right) and. Serial cultivated dilutions for 1 week. of cells were spotted onto the agar medium without terbinafine (left) and with 1 mM terbinafine (right) and cultivated for 1 week.

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Figure 3. (A) Sterols and (B) squalene content. Total lipid was extracted from log-phase cultures. All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05). ± Figure 3. Figure(A) Sterols 3. (A) Sterolsand (B and) squalene (B) squalene content. content. Total Total lipid lipid waswas extracted extracted from fr log-phaseom log-phase cultures. cultures. All data areAll meansdata areSD means ± SD of three biological replicates.Chlorophyll Significant differences and carotenoids between the wild are type major (WT) andphotosynthetic the mutants within pigments the same condition that are essential for of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condi- are indicated by asterisksphotoautotrophic (*) (p < 0.05). growth. Their synthesis also uses IPP as a precursor (Figure 1). Carote- tion are indicated by asterisks (*) (p < 0.05). noids also play a crucial role as antioxidants, preventing cell damage due to oxidative Chlorophyll and carotenoids are major photosynthetic pigments that are essential stressforChlorophyll [30]. photoautotrophic To investigate and carotenoids growth. the effects Their are of synthesis major increa photosyntheticsed also sterol uses IPP content as pigments a precursor on pigment that (Figure are content,1 essential). both for chlorophyllphotoautotrophicCarotenoids and also carotenoid growth. play a crucial Theirlevels role synthesis aswere antioxidants, measured also preventing uses in allIPP strains, cellas damagea precursor with due and to (Figure oxidativewithout 1). terbinaf- Carote- stress [30]. To investigate the effects of increased sterol content on pigment content, both inenoids being also added. play aIn crucial normal role medium, as antioxidants, the levels preventingof both carotenoid cell damage and chlorophyll due to oxidative were similarchlorophyll in all strains and carotenoid (Figure levels4A,B). were The measured only exception in all strains, was with the and carotenoid without terbinafine level of tfr3 that stressbeing [30]. added. To investigate In normal medium, the effects the levels of increa of bothsed carotenoid sterol andcontent chlorophyll on pigment were similar content, both waschlorophyll slightlyin all strains andlower (Figure carotenoid than4A,B). that Thelevelsof the only wereothers exception measured (Figure was the 4A). in carotenoid all Terbinafine strains, level with oftreatment andtfr3 that without was resulted terbinaf- in a decreaseine beingslightly in added. both lower carotenoid thanIn normal that of themedium,and others chlorophyll (Figurethe levels4 A).levels Terbinafineof both in the carotenoid WT treatment (Figure resultedand 4A,B). chlorophyll in aIn contrast, were bothsimilar decreasepigments in all instrains bothshowed carotenoid (Figure elevated 4A,B). and chlorophylllevels The onlyin all levels exception three in themutants WT was (Figure the when carotenoid4A,B). terbinafine In contrast, level was of tfr3 added that (Figureboth 4A,B). pigments showed elevated levels in all three mutants when terbinafine was added was (Figureslightly4A,B). lower than that of the others (Figure 4A). Terbinafine treatment resulted in a decrease in both carotenoid and chlorophyll levels in the WT (Figure 4A,B). In contrast, both pigments showed elevated levels in all three mutants when terbinafine was added (Figure 4A,B).

Figure 4. (A) Carotenoid and (B) chlorophyll content under N-replete medium in the presence and the absence of terbinafine. Figure 4. (A) Carotenoid and (B) chlorophyll content under N-replete medium in the presence and the absence of terbinaf- All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants ine. All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the within the same condition are indicated by asterisks (*) (p < 0.05). Significant differences between different treatments of the mutants within the same condition are indicated by asterisks (*) (p < 0.05). Significant differences between different treat- same strain are indicated by plus sign (+) (p < 0.05). ments of the same strain are indicated by plus sign (+) (p < 0.05). Figure 4. (A) Carotenoid and (B) chlorophyll content under N-replete medium in the presence and the absence of terbinafine. All data are means ± SD of Thethree overaccumulation biological replicates of. Significantsterols in Chlamydomonasdifferences between terbinafine-resistant the wild type (WT) mutants and the is in mutants within the same condition are indicated by asterisks (*) (p < 0.05). Significant differences between different treat- agreement with previous reports in yeast. The yeast Saccharomyces cerevisiae and Schizosac- ments of the same strain are indicated by plus sign (+) (p < 0.05). charomyces pombe terbinafine resistance mutants also exhibited higher ergosterol content The overaccumulation of sterols in Chlamydomonas terbinafine-resistant mutants is in agreement with previous reports in yeast. The yeast Saccharomyces cerevisiae and Schizosac- charomyces pombe terbinafine resistance mutants also exhibited higher ergosterol content

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The overaccumulation of sterols in Chlamydomonas terbinafine-resistant mutants is in agreement with previous reports in yeast. The yeast Saccharomyces cerevisiae and Schizosac- charomyces pombe terbinafine resistance mutants also exhibited higher ergosterol content than the wild-type cells [31,32]. Although the three mutants isolated in this work accumulated higher levels of sterol, only tfr1 accumulated higher level of squalene, whereas the other two mutants exhibited only half of the WT squalene level. This difference could be due to the differences in their mutations. The increase of both ergosterol and 7-dehydroporiferasterol was similar in all mutants. As there was no change in the ratio of these sterols, the mutations were unlikely to reside in the later steps of the pathway. The mutation in tfr1 might affect earlier steps, prior to squalene synthesis. An upregulation of the entire sterol pathway could lead to higher levels of intermediates upstream of squalene. By having more precursors for squalene synthesis, when squalene epoxidase enzyme is inhibited by terbinafine, this would result in a high level of squalene. On the other hand, the tfr2 and tfr3 mutants might have a desensitized squalene epoxidase or have high levels of the squalene epoxidase enzyme. The enzymes in the two mutants were able to convert more squalene into sterols, resulting in low squalene levels under terbinafine treatment. Nevertheless, evidence from the Schizosaccharomyces pombe mutant collection revealed that the mutations leading to terbinafine resistant phenotype could be in pathways other than squalene synthesis such as genes for mitochondrial function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and unknown genes [31]. Our previous work on terbinafine-sensitive mutants suggests that sensitivity to terbinafine could be used to screen for mutants that accumulate higher levels of triacylglycerol and carotenoids [29]. The mutants will be particularly useful for bringing microalgal biodiesel production closer to commercialization. All of the sensitive mutants, however, did not have higher levels of sterol. On the contrary, all of the terbinafine-resistant mutants in this work were found to produce higher levels of sterol. Therefore, it was clear that these two groups of mutants were affected in different pathways and could be used specifically for different purposes.

2.2. Effect of Environmental Stresses on Neutral Lipid and Pigments Cultivation of microalgae can be performed using water that is unsuitable for agricul- ture use such as brackish or industrial wastewater. Contaminants from wastewater and other environmental factors such as high light intensity, are known to induce oxidative stress [33,34]. In fact, tolerance to oxidative stress is a desirable property for algae grown in wastewater [35]. To investigate the phenotype of the WT and mutants under oxidative stress, growth on a TAP medium containing 80 mM NaCl and 4 µM Rose Bengal, a chemical that generates singlet oxygen, was tested. Only the tfr1 mutant exhibited greater tolerance to these chemicals compared to other strains (Figure5). Other than oxidative stress, nutrient stress is another factor that algae face in outdoor cultivation [36]. As microalgae have been recognized for their biotechnological potential in the production of biofuels in the past decade, many studies have reported how stresses, especially nutrient stress, lead to increased lipid accumulation [37–40]. Triacylglycerol or TAG is the type of lipid that can be converted to biodiesel via trans-esterification. The nitrogen-deprivation stress is one of the most widely used conditions for inducing the accumulation of TAG [33,41]. To investigate changes in lipid production after nitrogen deprivation, all strains were placed in a nitrogen-depleted medium with and without terbinafine. Nile Red dye was used to quantify neutral lipids based on the degree of fluorescence in each sample. The presence of terbinafine led to an increase in TAG content in all strains with the exception of tfr3 (Figure6A). Interestingly, nitrogen starvation combined with terbinafine caused a great increase in TAG production in tfr1 to about 2-fold compared to the WT level. In the case of pigments, the presence of terbinafine caused a decrease in the levels of both chlorophyll and carotenoids in the WT (Figure6B,C). Under terbinafine treatment, the levels of both pigments in tfr1 and tfr2 were significantly higher than in WT, as opposed to tfr3 that had lower levels of both pigments. For squalene, under Plants 2021, 10, 1673 6 of 13

nitrogen deficiency combined with terbinafine conditions, tfr1 and WT accumulated similar level of squalene (Figure6D). On the other hand, tfr2 and tfr3 still accumulated only 50% of the WT squalene level (Figure6D). Microalgae cultivated under environmental stress conditions such as nitrogen limita- tion, phosphorus deficiency, and high light intensity, are known to alter their lipid biosynthetic pathways towards the formation and accumulation of neutral lipids, mainly in the form of TAG [42,43]. The nitrogen starvation condition influences the distribution of carbon flux in microalgae [44]. Under this condition, degradation of the nitrogenous compounds such as proteins occurs, and carbon is diverted to storage compounds including lipids and carbohydrates [45]. The most interesting mutant was the tfr1 strain for its tolerance to environmental stresses and due to the high accumulation of TAG (Figures5 and6A) . The TAG level was also elevated in the presence of terbinafine, which induced an accumulation of squalene. Cells under terbinafine treatment also contained more carotenoids than the WT. These phenotypes supported the idea that the mutation in this mutant might lead to an upregulation of the sterol synthesis earlier in the pathway. When terbinafine was used Plants 2021, 10, x FOR PEER REVIEW to inhibit sterol synthesis, there might be higher levels of intermediates available for both 6 of 13

TAG and carotenoid synthesis. As sterols, squalene, and carotenoids possess antioxidant activities, the tfr1 was also able to withstand stresses better than other strains.

Figure 5. Growth phenotype wild type (WT) and tfr1 under oxidative stress. Serial dilutions of cells were spotted onto the agar medium NaCl and Rose Bengal (RB) and cultivated for 1 week. Figure 5. Growth phenotype wild type (WT) and tfr1 under oxidative stress. Serial dilutions of cells were spotted onto the agar medium NaCl and Rose Bengal (RB) and cultivated for 1 week.

Other than oxidative stress, nutrient stress is another factor that algae face in outdoor cultivation [36]. As microalgae have been recognized for their biotechnological potential in the production of biofuels in the past decade, many studies have reported how stresses, especially nutrient stress, lead to increased lipid accumulation [37–40]. Triacylglycerol or TAG is the type of lipid that can be converted to biodiesel via trans-esterification. The nitrogen-deprivation stress is one of the most widely used conditions for inducing the accumulation of TAG [33,41]. To investigate changes in lipid production after nitrogen deprivation, all strains were placed in a nitrogen-depleted medium with and without terbinafine. Nile Red dye was used to quantify neutral lipids based on the degree of fluorescence in each sample. The presence of terbinafine led to an increase in TAG content in all strains with the exception of tfr3 (Figure 6A). Interestingly, nitrogen starvation combined with terbinafine caused a great increase in TAG production in tfr1 to about 2-fold compared to the WT level. In the case of pigments, the presence of terbinafine caused a decrease in the levels of both chlorophyll and carotenoids in the WT (Figure 6B,C). Under terbinafine treatment, the levels of both pigments in tfr1 and tfr2 were significantly higher than in WT, as opposed to tfr3 that had lower levels of both pigments. For squalene, under nitrogen deficiency combined with terbinafine conditions, tfr1 and WT accumulated similar level of squalene (Figure 6D). On the other hand, tfr2 and tfr3 still accumulated only 50% of the WT squalene level (Figure 6D).

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FigureFigure 6.6. QuantificationQuantification of triacylglycerol triacylglycerol ( (AA),), chlorophyll chlorophyll (B (B),), carotenoid carotenoid (C (C) )and and squalene squalene (D (D) from) from cultures cultures under under N- N-depriveddeprived condition condition with with and and without without terbinafine. terbinafine. All Alldata data are aremeans means ± SD± ofSD three of three biological biological replicates. replicates. Significant Significant differ- differencesences between between the thewild wild type type (WT) (WT) and and the the mutants mutants within within the the same same condition condition are are indicated indicated by by asterisks (*) ((pp << 0.05).0.05). SignificantSignificant differences differences between between different different treatments treatments of of the the same same strain strain are are indicated indicated by by plus plus sign sign (+) (+) (p (p< < 0.05). 0.05).

2.3. GrowthMicroalgae and Photosynthetic cultivated under Efficiency environmental stress conditions such as nitrogen limi- tation,As phosphorustfr1 exhibited deficiency, the most promisingand high light phenotype, intensity, it wasare known further to investigated alter their lipid whether bio- orsynthetic not the pathways mutation withtowards regard the toformation this mutant and affectedaccumulation the efficiency of neutral of lipids, photosynthesis mainly in andthe form growth, of TAG both [42,43]. of which The will nitrogen affect product starvation yield. condition Under influences nitrogen-replete the distribution cultivation of conditions,carbon flux there in microalgae was no significant [44]. Under difference this condition, in cell growth degradation between of thethe WTnitrogenous and tfr1, withcom- orpounds without such terbinafine as proteins treatment occurs, and (Figure carbon7A,B). is diverted Light utilization to storage needs compounds to be efficient including in orderlipids to and maximize carbohydrates algal growth, [45]. The especially most interesting in outdoor mutant conditions, was the where tfr1 thestrain light for intensity its toler- changesance to environmental constantly. The stresses maximum and quantum due to th yielde high (Fv accumulation/Fm) of photosystem of TAG II(Figures (PSII) can 5 and be used6A). toThe monitor TAG level the efficiency was also ofelevated light utilization in the presence by the photosystemof terbinafine, II which (PSII) toinduced estimate an algalaccumulation health [46 of]. squalene. There was Cells essentially under terbin no differenceafine treatment in the Falsov/F containedm values betweenmore carote- the twonoids strains than the when WT. no These terbinafine phenotypes was addedsupported (Figure the 7ideaC). Inthat the the presence mutation of in terbinafine, this mutant however,might lead the to mutants an upregulation exhibited higher of the Fv /Fstermolvalues synthesis than theearlier WT, especiallyin the pathway. on later When days (Figureterbinafine7D). was Similarly, used to under inhibit nitrogen sterol synthesi starvations, there conditions, might be there higher was levels no difference of intermedi- in growthates available between for the both WT TAG and and the tfr1carotenoidmutant synthesis. (Figure8A,B). As sterols, However, squalene, unlike and nitrogen- carote- repletenoids possess condition, antioxidant nitrogen activities, deprivation the caused tfr1 was a substantialalso able to decreasewithstand in stresses the Fv/F betterm values than thatother never strains. fully recovered to the initial values, especially in the case of WT (Figure8C,D). The values of WT cultivated under nitrogen starvation conditions were lower than those of2.3. the Growth mutant and (Figure Photosynthetic8C). Agreat Efficiency drop in the WT F v/Fm values was observed under the combinationAs tfr1 exhibited of nitrogen the most starvation promising and phenotype, terbinafine, it whereas was further the valuesinvestigated of the whether mutant exhibitedor not the only mutation a small with drop regard (Figure to8 D).this Although mutant affected a significant the efficiency decrease of in photosynthesis F v/Fm values wasand observedgrowth, both especially of which under will nitrogen affect product depletion yield. condition, Under it nitrogen-replete does not directly cultivation translate toconditions, the same degreethere was of growthno significant inhibition. differ Thisence is in because cell growth Fv/F mbetweenmeasurement the WT isand rather tfr1, sensitive for determining the efficiency of PSII functions. Any decline in Fv/Fm values

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with or without terbinafine treatment (Figure 7A,B). Light utilization needs to be efficient in order to maximize algal growth, especially in outdoor conditions, where the light intensity changes constantly. The maximum quantum yield (Fv/Fm) of photosystem II (PSII) can be used to monitor the efficiency of light utilization by the photosystem II (PSII) to estimate algal health [46]. There was essentially no difference in the Fv/Fm values between the two strains when no terbinafine was added (Figure 7C). In the presence of terbinafine, however, the mutants exhibited higher Fv/Fm values than the WT, especially on later days Plants 2021, 10, 1673 8 of 13 (Figure 7D). Similarly, under nitrogen starvation conditions, there was no difference in growth between the WT and the tfr1 mutant (Figure 8A,B). However, unlike nitrogen- replete condition, nitrogen deprivation caused a substantial decrease in the Fv/Fm values canthat be never detected fully within recovered hours to of the stress initial treatments. values, especially The growth in the condition case of WT used (Figure here 8C,D). was mixotrophicThe values growth. of WT cultivated Therefore, under cells did nitrogen not rely starvation solely on conditions photosynthesis. were Theylower were than stillthose ableof tothe propagate mutant (Figure using the8C). carbon A great source drop provided in the WT in theFv/F medium.m values was As nitrogen observed starvation under the iscombination known to induce of nitrogen oxidative starvation stress, and and the terbin accumulationafine, whereas of TAG the has values also beenof the suggested mutant ex- ashibited a way to only relieve a small this drop stress (Figure by acting 8D). as Although an electron a sinksignificant [47,48], decrease the tfr1, within Fv/F them values ability towas produceobserved more especially TAG, was under able nitrogen to better toleratedepletion this co condition,ndition, it especiallydoes not directly with the translate addition to ofthe terbinafine. same degree of growth inhibition. This is because Fv/Fm measurement is rather sensi- tiveThe fortfr1 determiningstrain is a greatthe efficiency candidate of for PSII the functions. development Any of decline high value in F lipidv/Fm values production. can be Thedetected cost of within microalgal hours sterol of stress and squalenetreatments. production The growth can condition be lowered used as ahere result was of mixo- the abilitytrophic to overproducegrowth. Therefore, these cells lipids. did The not ability rely solely to withstand on photosynthesis. oxidative stressThey were induced still byable environmentalto propagate conditionsusing the carbon such as source fluctuating provided light orin contaminantsthe medium. As in waternitrogen in this starvation mutant is willknown result to in induce better oxidative growth and stress, higher and biomassthe accumulation yield. Furthermore, of TAG has thealso production been suggested of TAGas a for way biofuels to relieve was this also stress higher by inacting this mutant.as an electron The presence sink [47,48], of terbinafine the tfr1, with can the beused ability toto induce produce squalene more TAG, accumulation was able andto better reduce tolera contamination.te this condition, Therefore, especially several with growththe addi- conditionstion of terbinafine. can be applied to maximize the levels of desirable products.

Figure 7. Growth and maximum quantum yield (Fv/Fm) parameter of wild type (WT) and tfr1 mutant in nitrogen-replete medium (A,C) and nitrogen-replete medium with terbinaﬁne (B,D). All data are means ± SD of three biological replicates. Signiﬁcant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05). Plants 2021, 10, x FOR PEER REVIEW 9 of 13

Figure 7. Growth and maximum quantum yield (Fv/Fm) parameter of wild type (WT) and tfr1 mutant in nitrogen-replete Plantsmedium2021, 10, 1673(A,C) and nitrogen-replete medium with terbinafine (B,D). All data are means ± SD of three biological replicates.9 of 13 Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05).

Figure 8. Growth and maximum quantum yield (F /F ) parameter of wild type (WT) and tfr1 mutant in nitrogen-depleted Figure 8. Growth and maximum quantum yield (Fvv/Fmm) parameter of wild type (WT) and tfr1 mutant in nitrogen-depleted medium ((AA,,CC)) andand nitrogen-depletednitrogen-depleted medium medium with with terbinaﬁne terbinafine (B ,(DB,).D All). All data data are are means means± SD ± SD of threeof three biological biological replicates. replicates.Signiﬁcant Significant differences differences between between the wild the type wild (WT) type and (WT) the mutantsand the withinmutants the within same conditionthe same arecondition indicated are by indicated asterisks by (*) (asterisksp < 0.05). (*) (p < 0.05).

3. MaterialsThe tfr1 andstrain Methods is a great candidate for the development of high value lipid production.3.1.Culture The cost Conditions of microalgal and Mutant sterol Isolationand squalene production can be lowered as a result of the abilityC. reinhardtii to overproducewild-type these 4A+ lipids. strain The was ab providedility to withstand by Prof. Krishnaoxidative Niyogi stress (Univer-induced bysity environmental of California, Berkeley).conditions Exponential-phasesuch as fluctuating cellslight at or a contaminants density of 5 × in10 water6 cells in mL this−1 mutantwere diluted will result to 2 in× better106 cells growth mL and−1 in higher fresh biomass Tris-acetate-phosphate yield. Furthermore, (TAP) the mediumproduction or ofTAP-N TAG mediumfor biofuels as nitrogen-repletewas also higher in or this nitrogen-deplete mutant. The presence conditions, of terbinafine respectively. can Cul- be usedtures to were induce placed squalene on an orbitalaccumulation shaker atand 120 reduce rpm, 25 contamination.◦C with constant Therefore, illumination several at growth50 µmol conditions photons m can−2 sbe−1 .appliedTerbinafine, to maximize NaCl, orthe Rose levels Bengal of desirable was added products. to warm TAP agar to prepare plates containing each chemical. For terbinafine treatment, terbinafine was 3.added Materials to the and culture Methods to a final concentration of 10 µM. Cells were harvested by centrifuga- ◦ 3.1.tion Culture at 6000 Conditions rpm for 10 and min Mutant and kept Isolation at −80 C until use. C. reinhardtii × 6 C.To reinhardtii generatea wild-type mutant population, 4A+ strain culturewas provided of by Prof. atKrishna a density Niyogi of 5 (University10 cells mL−1 was placed in a Petri dish and kept in darkness for 1 h. The culture was irradiated of California, Berkeley). Exponential-phase cells at a density of 5 × 106 cells mL−1 were with UV light for 30 min using a UV transilluminator (Model M-26, Upland, CA, USA). diluted to 2 × 106 cells mL−1 in fresh Tris-acetate-phosphate (TAP) medium or TAP-N me- This treatment resulted in a 0.1–0.2% survival rate as previously determined. Following dium as nitrogen-replete or nitrogen-deplete conditions, respectively. Cultures were an overnight incubation in the dark, cells were placed onto TAP medium plates and incubated at 30–35 µmol photons m−2 s−1 at 25 ◦C. After two weeks, visible colonies were patched onto fresh TAP medium plates. A total of 1,920 colonies were obtained. After the patches turned dark green, they were replica plated onto TAP medium plates containing

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0.1–1.0 mM of terbinaﬁne to identify strains that exhibited greater resistance to terbinaﬁne than the parental strain.

3.2. Analysis of Sterols, Squalene, and Tag Content Total lipid was extracted from 8 × 107 cells following the method recommended by [49]. Cells were mixed with 4 mL chloroform:methanol (2:1, v/v) and shaken until the biomass was dispersed in the solvent system [50]. One milliliter of 0.9% NaCl was added and the tube was vortexed to mix. After centrifugation, a pasture pipette was used to collect the bottom layer and place the liquid into a new tube. It was then dried under nitrogen gas and kept at −80 ◦C until use. Squalene was quantified by high-performance liquid chromatography (HPLC). Total lipid was dissolved in 250 µL of acetonitrile and filtered through a 0.2 µm PTFE filter. HPLC was performed using the Waters HPLC system and a C18 column (150 × 3.0 mm, 5 µm particle size, Phenomenex, CA, USA). The mobile phase was 100% acetonitrile with a flow rate of 1.5 mL min−1. Squalene was detected at 195 nm and was quantified using a previously generated standard curve of peak area vs. known squalene amount. Sterol content was determined as previously described [29,51]. The sample was analyzed using gas chromatography–mass spectrometry (GCMS-QP20202; Shimadzu, Kyoto, Japan) equipped with a DB-5MS capillary column (Agilent Technologies, Santa Clara, CA, USA), carrier gas: He (1 mL min−1); oven temperature: 150–300 ◦C (increase rate 20 ◦C min−1). The ionization voltage was 70 eV, and the scan range was 40–500 Da. The ergosterol and 7- dehydroporiferaseterol peaks were identified by their respective standards (Sigma-Aldrich, Munich, Germany), and the contents were compared by peak areas. A relative level of triacylglycerol was quantified using Nile Red fluorescence [52]. In brief, cells were diluted to a density of 2 × 106 cells mL−1. Then, 50 µL of dimethyl sulfoxide and 2.5 µL of Nile Red were added to a microtube containing 950 µL of culture. Samples were gently mixed and kept in the dark for 10 min. Two-hundred microliters of each sample was transferred to a 96-well plate. Fluorescent intensity was measured in arbitrary units by a Microplate Reader (TECAN SparkControl v1.1.13.0, Zurich, Switzerland) using the excitation/emission wavelength at 528/576 nm.

3.3. Growth, Pigment Quantiﬁcation, and Photosynthetic Parameter Cell growth was determined by counting cells on a hemacytometer under a microscope. The photosynthetic pigments were extracted from 1 mL of culture using 1 mL of 80% acetone. The quantity of chlorophyll and carotenoid was calculated following a previously reported formula [53]. The photosynthesis efﬁciency expressed by the ratio of Fv/Fm was determined using AquaPen AP100 (Photon systems instrument, Drasov, Czech Republic).

4. Conclusions Resistance to terbinafine in green microalgae such as Chlamydomonas as noted in this work, can lead to an overaccumulation of sterols and squalene. It is possible to isolate those mutants that also accumulate other high value products such as pigments and TAG. Tolerance to environmental stresses in such strains allows better photosynthetic efficiency and growth performance. If needed, the presence of terbinafine can help eliminate unwanted contamination. Our data offer a novel solution for improving productivity of high value lipids in green microalgae.

Author Contributions: Conceptualization, S.P., S.J. and A.S.; methodology, S.P., S.J., N.S., and S.V.; formal analysis, S.P., S.J. and A.S.; investigation, S.P., S.J. and A.S.; writing—original draft preparation, S.P. and A.S.; writing—review and editing, A.S., supervision, A.S., project administration, A.S., funding acquisition, A.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by International SciKU Branding (ISB) fund and Basic Research Fund (BRF), Faculty of Science, Kasetsart University, Kasetsart University Research and Development Plants 2021, 10, 1673 11 of 13

Institute (KURDI), and Thailand Research Fund (RSA6080030). This research was also funded by Kasetsart University through the Graduate School Fellowship Program. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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