| Summary: | Lichens are a natural source of bioactive compounds. <i>Cladonia metacorallifera</i> var. <i>reagens</i> KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, thamnolic, 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 <i>C. metacorallifera</i> 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 after 3 weeks of culture, implying that these genes may be required for cristazarin production in <i>C. metacorallifera.</i> Moreover, the white collar genes <i>Cmwc-1</i> and <i>Cmwc-2</i> were highly upregulated at all times under light conditions, indicating a possible correlation between cristazarin production and gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC<sub>50</sub> values of 18.2, 26.1, and 30.9 μg/mL, respectively, which highlights the value of cristazarin. Overall, our results suggest that 1% fructose under light conditions is required for cristazarin production by <i>C. metacorallifera</i> mycobionts, and cristazarin could be a good bioactive compound.
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