The spontaneous solid-state fermentation of high-temperature Daqu (HTD) is a temperature-dependent stacking bioprocessing for enriching microbiota and enzymes to guarantee efficient substrate utilization and fermentation. However, there is a lack of clarity regarding how temperature adaptability affects HTD microbial assembly, domestication direction, and metabolic profile. Here, the flavor substances, microbial assembly, metabolic network, and horizontal gene transfer (HGT) events of three HTDs from Renshu (RS), Jiushang (JS), and Maoyuan (MY) were analyzed. 125 volatile compounds were identified, tetramethylpyrazine, 3-methyl-butanoic acid, phenylethyl alcohol, and trimethylpyrazine were clarified as the typical flavor substances. Bacillus and Kroppenstedtia were the shared dominant bacterial genera. Paecilomyces, Aspergillus, Rasamsonia, and Lichtheimia were dominant fungal genera. Differences in flavor metabolism, microbial structure, and key enzyme metabolism are strongly correlated with sample distance. As proximity decreases, the microbial structural and functional metabolic traits tend to exhibit greater similarity. The frequency of HGT events was analyzed using MetaCHIP, 49, 9 and 69 groups of HGT events occurred in RS, JS, and MY, respectively. HGT events occurred most abundantly in Bacillaceae, and the microbial taxa with a closer phylogenetic relationship possessed the highest incidence of HGT. Specifically, the occurrence of HGT was mainly associated with high-temperature adaptability. It was also linked to characteristic flavor metabolism. Our results revealed the effects of temperature stress on microbial regulation of HTD and adaptive transfer of relevant genes in stacked fermented HTDs. This work provides important insights into HTD quality classification and regulation of solid-state fermentation quality and efficiency through microbial domestication.

Keywords: High-temperature Daqu; Horizontal gene transfer; Metagenome binning; Microbial assembly; Solid-state fermentation; Temperature adaptability.

Copyright © 2025. Published by Elsevier Ltd.

高温大曲（HTD）的自发固态发酵是一种温度依赖性的堆叠生物处理过程，用于丰富微生物群和酶，以保证高效的底物利用和发酵。然而，关于温度适应性如何影响HTD微生物组成、驯化方向及代谢谱尚不清楚。本文分析了来自任薯（RS）、九酿（JS）和茅源（MY）的三种高温大曲中的风味物质、微生物组成、代谢网络以及水平基因转移（HGT）事件。共鉴定出125种挥发性化合物，四甲基吡嗪、3-甲基丁酸、苯乙醇和三甲基吡嗪被确认为典型的风味物质。芽孢杆菌和克罗彭施泰特菌是共享的主要细菌属。皮青霉、曲霉、拉斯蒙森氏酵母和利希体菌为主要真菌属。风味代谢、微生物结构及关键酶代谢与样品距离强相关，随着距离的减小，微生物结构和功能代谢特征表现出更高的相似性。使用MetaCHIP分析HGT事件频率，在RS、JS和MY中分别发生了49组、9组和69组HGT事件。HGT事件在芽孢杆菌科中最丰富，并且具有更近亲缘关系的微生物类群拥有最高的HGT发生率。具体而言，HGT的发生主要与高温适应性相关，同时也与其特征风味代谢有关。我们的研究结果揭示了温度压力对HTD中微生物调节的影响以及堆叠发酵HTD中相关基因适应性转移的效果。这项工作为通过微生物驯化来分类和调控固态发酵质量提供了重要见解。

关键词：
高温大曲；水平基因转移；元基因组比对；微生物组成；固态发酵；温度适应性。

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