Fermented soybeans are popular among many for their rich soy sauce-like aroma. However, the precise composition of this aroma remains elusive, with key aroma compounds unidentified. In this study, we screened the candidate genes ilvA and serA in BJ3-2 based on previous multi-omics data, and we constructed three mutant strains, BJ3-2-ΔserA, BJ3-2-ΔilvA, and BJ3-2-ΔserAΔilvA, using homologous recombination to fermented soybeans with varying intensities of soy sauce-like aroma. Our objective was to analyze samples that exhibited different aroma intensities resulting from the fermented soybeans of BJ3-2 and its mutant strains, thereby exploring the key flavor compounds influencing soy sauce-like aroma as well analyzing the effects of ilvA and serA on soy sauce-like aroma. We employed quantitative descriptive sensory analysis (QDA), gas chromatography–olfactometry–mass spectrometry (GC-O-MS), relative odor activity value analysis (rOAV), principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and partial least squares regression analysis (PLSR). QDA revealed the predominant soy sauce-like aroma profile of roasted and smoky aromas. GC-MS detected 99 volatile components, predominantly pyrazines and ketones, across the four samples, each showing varying concentrations. Based on rOAV (>1) and GC-O, 12 compounds emerged as primary contributors to soy sauce-like aroma. PCA and OPLS-DA were instrumental in discerning aroma differences among the samples, identifying five compounds with VIP > 1 as key marker compounds influencing soy sauce-like aroma intensity levels. Differential analyses of key aroma compounds indicated that the mutant strains of ilvA and serA affected soy sauce-like aroma mainly by affecting pyrazines. PLSR analysis indicated that roasted and smoky aromas were the two most important sensory attributes of soy sauce-like aroma, with pyrazines associated with roasted aroma and guaiacol associated with smoky aroma. In addition, substances positively correlated with the intensity of soy sauce-like aroma were verified by additional experiments. This study enhances our understanding of the characteristic flavor compounds in soy sauce-like aroma ferments, provides new perspectives for analyzing the molecular mechanisms of soy sauce-like aroma formation, and provides a theoretical framework for the targeted enhancement of soy sauce-like aroma in various foods.
