Journal of Fungi Article Production and Activity of Cristazarin in the Lichen-Forming Fungus Cladonia metacorallifera Min-Hye Jeong 1,2,†, Chan-Ho Park 1,† , Jung A Kim 3,†, Eu Ddeum Choi 2, Soonok Kim 3, Jae-Seoun Hur 1,* and Sook-Young Park 2,* 1 Korean Lichen Research Institute, Sunchon National University, Sunchoeon 57922, Korea; [email protected] (M.-H.J.); [email protected] (C.-H.P.) 2 Department of Plant Medicine, Sunchon National University, Suncheon 57922, Korea; [email protected] 3 National Institute of Biological Resources, Incheon 22689, Korea; [email protected] (J.A.K.); [email protected] (S.K.) * Correspondence: [email protected] (J.-S.H.); [email protected] (S.-Y.P.); Tel.: +82-61-750-3383 (J.-S.H.); +82-61-750-3868 (S.-Y.P.) † These authors contributed equally to this work. Abstract: Lichens are a natural source of bioactive compounds. Cladonia metacorallifera var. reagens KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, tham- nolic, and didymic acids. However, these metabolites have not been detected in isolated mycobionts. We investigated the effects of six carbon sources on metabolite biosynthesis in the C. metacorallifera mycobiont. Red pigments appeared only in Lilly and Barnett’s media with fructose at 15 ◦C after 3 weeks of culture and decreased after 6 weeks. We purified these red pigments using preparative- scale high performance liquid chromatography and analyzed them via nuclear magnetic resonance. Results indicated that 1% fructose-induced cristazarin and 6-methylcristazarin production under light conditions. In total, 27 out of 30 putative polyketide synthase genes were differentially expressed Citation: Jeong, M.-H.; Park, C.-H.; after 3 weeks of culture, implying that these genes may be required for cristazarin production in Kim, J.A; Choi, E.D.; Kim, S.; Hur, C. metacorallifera. Moreover, the white collar genes Cmwc-1 and Cmwc-2 were highly upregulated at J.-S.; Park, S.-Y. Production and Activity of Cristazarin in the all times under light conditions, indicating a possible correlation between cristazarin production and Lichen-Forming Fungus Cladonia gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC50 metacorallifera. J. Fungi 2021, 7, 601. values of 18.2, 26.1, and 30.9 µg/mL, respectively, which highlights the value of cristazarin. Overall, https://doi.org/10.3390/jof7080601 our results suggest that 1% fructose under light conditions is required for cristazarin production by C. metacorallifera mycobionts, and cristazarin could be a good bioactive compound. Academic Editor: Laurent Dufossé Keywords: Cladonia metacorallifera; cristazarin; lichen bioresource; polyketide synthase (PKS) genes; Received: 30 June 2021 secondary metabolites Accepted: 21 July 2021 Published: 26 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Lichens are symbiotic organisms composed of a lichen-forming fungus (the mycobiont) published maps and institutional affil- and an alga, a cyanobacterium, or both (the photobiont). Lichens produce various charac- iations. teristic secondary metabolites, such as depsides, depsidones, dibenzofurans, pulvinates, chromones, and quinones. Notably, these metabolites, which have been detected in extracts of lichen thalli, are also produced when isolated mycobionts are cultured without their algal partners [1–4]. However, some mycobiont isolates cannot produce the metabolites Copyright: © 2021 by the authors. that are detected in their symbiotic lichen form [5,6]. While the precise causal factors are Licensee MDPI, Basel, Switzerland. unknown, it is expected that differences in nutrient conditions, especially carbon sources, This article is an open access article account for changes in the polyketide biosynthesis pathway, which lead to differences in distributed under the terms and secondary metabolite profiles [7]. The secondary metabolite productions in various lichens conditions of the Creative Commons Attribution (CC BY) license (https:// with different carbohydrate carbon sources have been compared [8–11]. Lichen mycobionts creativecommons.org/licenses/by/ have been reported to produce secondary metabolites in culture media supplemented with 4.0/). 10–20% sucrose [9–12], mannitol, or ribitol [8]. J. Fungi 2021, 7, 601. https://doi.org/10.3390/jof7080601 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, x FOR PEER REVIEW 2 of 15 J. Fungi 2021, 7, 601 2 of 14 Lichen mycobionts have been reported to produce secondary metabolites in culture media supplementedThe red compounds with 10–20% cristazarin sucrose [9 (3-ethyl-2-hydroxy-7-methoxynaphthazarin)–12], mannitol, or ribitol [8]. and 6- methylcristazarinThe red compounds are derived cristazarin from (3 naphthazarin-ethyl-2-hydroxy (5,8-dihydroxy-1,4-naphthoquinone;-7-methoxynaphthazarin) and 6- Figuremethylcristazarin1 ) and produced are derived by the isolatedfrom naphthazarinCladonia cristatella (5,8-dihydroxymycobiont-1,4 [6-].naphthoquinone; These compounds haveFigure been 1) and identified produced in solid by the or liquidisolated cultures Cladonia of cristatella the C. cristatella mycobiontmycobiont [6]. These [6 ,com-12], but pounds have been identified in solid or liquid cultures of the C. cristatella mycobiont [6,12], not in the lichen thalli of C. cristatella nor in other lichens to date. Furthermore, cristazarin but not in the lichen thalli of C. cristatella nor in other lichens to date. Furthermore, cris- displays potent biological properties, including antibacterial and antitumor activities [7], tazarin displays potent biological properties, including antibacterial and antitumor activ- and hence may be a potential antibacterial and antitumor agent. The red compounds ities [7], and hence may be a potential antibacterial and antitumor agent. The red com- cristazarin (3-ethyl-2-hydroxy-7-methoxynaphthazarin) and 6-methylcristazarin are de- pounds cristazarin (3-ethyl-2-hydroxy-7-methoxynaphthazarin) and 6-methylcristazarin rived from naphthazarin (5,8-dihydroxy-1,4-naphthoquinone; Figure1) and produced by are derived from naphthazarin (5,8-dihydroxy-1,4-naphthoquinone; Figure 1) and pro- the isolated Cladonia cristatella mycobiont [6]. These compounds have been identified in duced by the isolated Cladonia cristatella mycobiont [6]. These compounds have been iden- C. cristatella solidtified or in liquid solid or cultures liquid cultures of the of the C. cristatellamycobiont myco [6biont,12], [6,12] but not, but in not the in lichen the lichen thalli of C.thalli cristatella of C. cristatellanor in other nor in lichens other lichens to date. to Furthermore, date. Furthermore, cristazarin cristazarin displays displays potent potent biolog- icalbiological properties, properties, including including antibacterial antibacterial and and antitumor antitumor activities activities [7], [7] and, and hence hence may may be a potentialbe a potential antibacterial antibacterial and and antitumor antitumor agent agent FigureFigure 1.1. ChemicalChemical structure of of naphthazarin, naphthazarin, cristazarin, cristazarin, and and 6-methylcristazarin. 6-methylcristazarin. TheThe majoritymajority of secondary metabolites metabolites in in fungi fungi and and lichens lichens are are aromatic aromatic polyketides, polyketides, suchsuch asas cristazarincristazarin [[13]13].. Polyketide Polyketide metabolites metabolites constitute constitute a structurally a structurally diverse diverse family family of of naturalnatural compounds.compounds. They are are produced produced by by the the successive successive condensation condensation of acyl of acyl coenzyme coenzyme AA (CoA) (CoA) subunits subunits in in a a head-to-tail head-to-tail manner manner viavia thethe polyketidepolyketide pathwaypathway [[14,15]14,15],, andand theythey are synthesizedare synthesized by polyketide by polyketide synthases synthases (PKSs). (PKSs). In recentIn recent years, years, several several advanced advanced techniques, tech- includingniques, including whole-genome whole-genome sequencing sequ andencing bioinformatics, and bioinformatics, have offered have offered new opportunities new op- toportunities identify putative to identify PKS putative genes PKS via knowngenes via domains known suchdomains as the such keto-synthase as the keto-synthase (KS), acyl- transferase(KS), acyltransferase (AT), and (AT), acyl carrier and acyl (AC), carrier and (AC), to predict and theto predict putative the chemical putative structureschemical of theirstructures corresponding of their corresponding secondary metabolites secondary without metabolites genetic without nor chemical genetic analysesnor chemical [16– 24]. Moreover,analyses [16 the–24 analysis]. Moreover, of gene the expressionanalysis of providesgene expression a comprehensive provides a comprehensive understanding of geneunderstanding regulation of under gene specificregulation conditions. under specific conditions. Light is a major environmental signal that influences various biological activities. In Light is a major environmental signal that influences various biological activities. In fungi, light is a source of information, such as the induction or inhibition of morphogene- fungi, light is a source of information, such as the induction or inhibition of morphogenesis, sis, reproduction, phototrophy, circadian clock resetting, and secondary metabolism [25– reproduction, phototrophy, circadian clock resetting, and secondary metabolism [25–30]. 30]. Furthermore, the production