AbstractBACKGROUNDThere are few reports on the breeding of high‐yielding tetramethylpyrazine (TTMP) strains in strong‐flavor Daqu. In addition, studies on the mechanism of TTMP production in strains are mostly based on common physiological and biochemical indicators, and there is no report on RNA level. Therefore, in this study, a strain with high production of TTMP was screened out from strong‐flavor liquor, and transcriptome sequencing analysis was performed to analyze its key metabolic pathways and key genes, and to infer the mechanism of TTMP production in the strain.RESULTSIn this study, a strain with a high yield of tetramethylpyrazine (TTMP) was screened out, and the yield was 29.83 μg mL−1. The identified strain was Bacillus velezensis, which could increase the content of TTMP in liquor by about 88%. After transcriptome sequencing, a total of 1851 differentially expressed genes were screened, including 1055 up‐regulated genes and 796 down‐regulated genes. Three pathways related to the production of TTMP were identified by gene ontology (GO) annotation and COG annotation, including carbohydrate metabolism, cell movement and amino acid metabolism. The key genes of TTMP were analyzed, and the factors that might regulate the production of TTMP, such as the transfer of uracil phosphate ribose and glycosyltransferase, were obtained.CONCLUSIONSA strain of B. velezensis with high TTMP production was screened and identified in strong‐flavor Daqu for the first time. The yield of TTMP was 29.83 μg mL−1, which increased the TTMP content in liquor by 88%. The key metabolic pathways of TTMP production in the strain were obtained: carbohydrate metabolism, cell movement and amino acid metabolism, and the key regulatory genes of each pathway were found, which complemented the gap in gene level in the production regulation of the strain, and provided a theoretical basis for the subsequent study of TTMP in liquor. © 2023 Society of Chemical Industry.