Rapid Phenotypic and Metabolomic Domestication of Wild Penicillium Molds on Cheese

Experimental evolution of Penicillium on cheese. (A) Evolution of Penicillium commune strain 162_3FA on cheese curd alone (“Penicillium alone”) and in the presence of three competing cheese rind microbes (“Penicillium + community”; Staphylococcus xylosus, Brachybacterium alimentarium, and Debaryomyces hansenii). Lines connect points representing mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. “Penicillium + community” had a significantly lower domesticated phenotype frequency (repeated-measures ANOVA; see text for statistics). (B) Experimental evolution of P. commune strain 162_3FA in different cheese nutrient environments. “Normal cheese” = 10% cheese curd in agar medium. “Low cheese” = 1% cheese curd in agar medium. “Alternating normal/low” = alternating 10% and 1% cheese curd at each transfer. Both “Low cheese” and “Alternating normal/low” had significantly lower domesticated phenotype frequencies (repeated-measures ANOVA with Tukey’s HSD post hoc tests; see text for statistics). Lines connect points representing mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. The “low cheese” line is difficult to see because it is at 0%. (C) Morphology of four representative colony types. The ancestor phenotype was dark blue-green and dusty; domesticated phenotype 1 was white and flat; domesticated phenotype 2 was white and fuzzy/dusty; domesticated phenotype 3 was blue-green but had less intense coloration than the ancestor and a less fuzzy appearance. (D) Reproductive output and cyclopiazonic acid production of a range of strains isolated across the experimental evolution populations. Points are mean values, and error bars represent 1 standard deviation of the mean. Strains M5, M6, M7, and M8 had reduced reproductive compared to the ancestor (Dunnett’s test, P < 0.05). All domesticated phenotypes had significantly reduced CPA production compared to the ancestor (Dunnett’s test, P < 0.05). (E) Competition between ancestral P. commune strain 162_3FA and domesticated strain M9. Strain M9 outcompeted the ancestor after 10 days of growth on cheese curd agar. Points represent individual replicate competition communities, and the horizontal line indicates mean values. Error bars represent 1 standard deviation.

Volatile organic compound (VOC) production of ancestral and domesticated Penicillium. Because total concentrations of VOCs are highly variable across different compounds, visualization was simplified by relativizing the relative peak areas from GC-MS chromatograms within each VOC to the highest concentration detected for that VOC. Only the VOCs that were detected across all replicates are shown. See Table S2 for all VOCs and their relative peak area values. The UPGMA tree is clustering the VOC profiles for each replicate based on Bray-Curtis dissimilarity. Asterisks indicate clusters with >70% bootstrap support. WT, ancestor phenotype. M2, M5, and M6 are all domesticated phenotypes. Numbers 1 to 4 after strain designations indicate biological replicates. Because of accidental sample loss during processing, only three biological replicates were collected from the WT. Descriptors on the right represent known aroma qualities of detected VOCs.

Experimental domestication shifts global gene expression of Penicillium on cheese. (A) Differences in gene expression between ancestor and domesticated Penicillium commune 162_3FA. Each dot represents a transcript from across the genome. Yellow dots represent those transcripts that had higher expression, and blue dots represent those transcripts that had lower expression (5-fold change in expression; FDR-corrected P value < 0.05). (B) Pathway enrichment analysis showing the distribution of GO terms that were significantly enriched in genes with decreased expression in the domesticated phenotype (strain M5). (C) Representative mapping of reads to the ergot alkaloid synthesis (eas) gene cluster.