Objective To explore the key non-ribosomal peptide synthetase (NRPS) genes of the active peptide Asp-Leu-Ala-Pro (DLAP) with angiotensin-converting enzyme (ACE) inhibitory activity identified from the mycelia of Ganoderma sinense.Methods The effects of key NRPS genes on the synthesis of DLAP are analyzed by PEG-mediated genetic transformation experiment, real-time fluorescence quantitative PCR (qRT-PCR), and high performance liquid chromatography (HPLC).Results qRT-PCR results reveal that compared with the wild type, the silenced transformants of GSI_00900 have the reductions of 44.99%, 68.88%, and 66.17% in the expression level of this gene. The three silenced transformants of GSI_10299 show the reductions of 74.11%, 78.01%, and 75.76% in the expression level of this gene. The two silenced transformants of GSI_10830 present reductions of 30.55% and 76.99%, and those of GSI_11441 exhibit the expression declines ranging from 19.43% to 84.80%. HPLC results indicate that the DLAP content in each positive transformant was lower than that in the wild type (P<0.05).Conclusion These four NRPS genes (GSI_00900, GSI_10299, GSI_10830, and GSI_11441) play a crucial role in the synthesis of DLAP.