| Summary: | Abstract Aroma is an important factor affecting mushroom character and quality. According to the different reaction pathway, the key aroma metabolites (sulfur and eight‐carbon volatiles) formation can be classified into enzymatic reactions and nonenzymatic reactions. Aroma volatiles are generated from precursors via the biocatalytic activities of various synthases during the growth stages of shiitake mushrooms. Understanding the specific relationships between the key aroma metabolites and their synthases is key to improving shiitake mushroom quality. At the same time, to reduce forest logging and burning of agricultural by‐products in farmland, agricultural by‐products have been applied to shiitake mushroom cultivation. Nevertheless, how to further improve the production of aroma volatiles in mushroom cultivated with agricultural waste is still a challenge. In order to understand the biosynthesis of volatiles via enzymatic reactions and screen the agricultural by‐products that can improve the production of aroma volatiles in mushroom cultivation, the mechanism of producing aroma volatiles needs to be further elucidated. In this study, the activities and gene expression levels of the key synthases involved in volatile metabolism, the contents of key aroma volatiles, and the correlations between related synthetase, volatiles, and cultivation substrate (CS) were investigated. Network models for visualizing the links between synthetase, volatiles, and CSs were built through partial least squares (PLS) regression analysis. The correlation coefficients among three related synthetase and enzymatic gene expression were high, and the combined effects of multiple synthetase promoted the production of volatiles. PLS analysis showed that the corncob and corn meal were more related to the production of volatiles and synthetase gene expression, and they can be added to the CSs as flavor promoting substances. The enrichment of key aroma volatiles in shiitake mushroom cultivated by the gradient of 20% corn meal combination CS was noticeable.
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