Evaluation and transcriptomic and metabolomic analysis of the ability of Auricularia heimuer to utilize crop straw

Abstract

Auricularia heimuer is an important edible fungus, and the choice of its cultivation medium is very important to improve the yield and quality. Traditionally, A. heimuer mostly uses wood chips as cultivation substrate, but with the increase of agricultural waste, exploring agricultural straw as an alternative substrate has become a research hotspot. In this study, a wild A. heimuer strain W-ZD22 with good adaptability to straw matrix was used to measure mycelia growth characteristics and extracellular enzyme activity. Transcriptomics and non-targeted metabolomics methods were used to compare the effects of mycelia using agricultural straw matrix and wood chips matrix. It was found that the lignin enzyme activities of corn straw and wood chips were similar. By transcriptomic and metabolomic analysis, we further analyzed the transcription profiles of A. heimuer mycelia grown in different substrates (straw and corn stalk, straw and sawdust, corn stalk and sawdust), and identified 5,149, 2,740 and 2,933 different expression genes (DEGs), respectively. The three control groups had a total of 526 gene variants. The top 20 pathways with the highest concentration of DEGs mainly involved glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, oxidative phosphorylation, endoplasmic reticulum protein processing and ribosome. In order to further understand the similarity of enzyme activity of Auricularia mycelium on corn stalk and wood chips, metabolomic analysis of substrate of corn stalk and wood chips was conducted. It was found that different metabolites were significantly enriched in starch and sucrose metabolism, glutathione metabolism, carbon metabolism and other pathways, which provided theoretical basis for efficient comprehensive utilization of corn stalk in auricularia growth.

Keywords: Auricularia heimuer; Crop straw; Metabolomics; Transcriptomics.

Figure 1. Morphology of fruiting body of wild Auricularia heimuer.

Test materials.

Figure 2. Image of adaptability and colony morphology characteristics of mycelial straw on the 8th day.

Strain culture characteristics: 25 °C cultivation is optimal, the age is 10~12 days, neat colony edges, and strong mycelial vitality. D1 is rice straw, D2 is rice husk, D3 is corn stalk, D4 is corn cob, D5 is bean stalk, D6 is sawdust.

Figure 3. (A–D) Changes in cellulase activity.

Note: FPA is total cellulase, C₁ is endoglucanase, C_x is circumscribed glucanase, β-G is β-D-Glucosidase. D1 is rice straw, D2 is rice husk, D3 is corn stalk, D4 is corn cob, D5 is bean stalk, D6 is sawdust. Letters a, b, c, d and e indicate significant differences, while the same letters indicate no significant differences.

Figure 4. Changes in activities of hemicellulase and ligninase.

Note: D1 is rice straw, D2 is rice husk, D3 is corn stalk, D4 is corn cob, D5 is bean stalk, D6 is sawdust. Letters a, b, c, d and e indicate significant differences, while the same letters indicate no significant differences.

Figure 5. Correlation heatmap between different substrates, extracellular enzyme activity, and mycelial growth rate.

Red indicates a positive correlation; green indicates negative correlation. The shade of color indicates the degree of correlation. The asterisk (*) represents significant correlation. C(%) represents carbon content; N(%) represents nitrogen content; C/N is the carbon-to-nitrogen ratio; Cellulose is the content of cellulose; Hemicellulose is the content of hemicellulose; Ligin is the content of lignin; Cellulose/Lignin ratio is the ratio of cellulose to lignin content; FPA is the total cellulase activity; C1 is the endo-glucanase activity; CX is the exo-glucanase activity; β-G is the β-glucosidase activity; Xylanase is the xylanase activity; Lip is the peroxidase activity; MnP is the manganese peroxidase activity; Lac is the laccase activity; Mycelial growth rate is the growth rate of mycelium.

Figure 6. Analyzes the differentially expressed genes (DEG) occurring in the three comparison groups.

(A) Stacked bar chart of the number of DEGs in different comparisons. (B) The Venn diagram of overlapping DEG in the three comparison groups. (C) Hierarchical clustering heatmap of 526 DEGs common in the three comparison groups. Red represents high-expressed genes and green represents low-expressed genes

Figure 7. Gene Ontology (GO) and KEGG annotations of DEG.

(A) The GO term with the highest degree of DEG enrichment among the three comparison groups; (B) the KEGG Pathway with the highest degree of DEG enrichment among the three comparison groups; (C) bubble diagram of carbohydrate metabolism pathways.

Figure 8. PCA and OPLS-DA analysis of non-targeted metabolomics.

Samples PCA scores of non-targeted metabolomics under positive ion mode (A) and negative ion mode (B); (B) OPLS-DA model establishment and substitution test results.

Figure 9. Screening of differential metabolites.

(A) Histogram of differential metabolism in MERGE mode; (B) heatmap of differential accumulation metabolites; (C) top 20 differential metabolites.

Figure 10. Enrichment of the top 20 KEGG differential metabolic pathways in D3 vs D6.

Figure 11. KEGG enrichment analysis of differential metabolites and individual omics of differential genes.

(A) Bar chart of metabolic pathways by cation mode integrated analysis. (B) Bar chart of metabolic pathways by anion mode integrated analysis. (C) Pathway map of differential metabolites and differential transcripts in starch and sucrose metabolism. The x-axis of the bar chart represents metabolic pathways, the blue bar represents the enriched p-value of differential genes, and the red bar represents the enriched p-value of differential metabolites, represented by −log (p-value). The larger the y-axis, the stronger the significant enrichment degree. Note: α-glucosidase (EC3.2.1.20), α-trehalose glucose hydrolase (EC3.2.1.28), α-amylase (EC3.2.1.1), β-glucosidase (EC3.2.1.21), UDP-glucose pyrophosphorylase (EC2.7.7.9), glycogen phosphorylase (EC2.4.1). 1), glycogen synthetase (EC2.4.1.11), carboxyester hydrolase (EC3.1.3.12).